ill 


/*#* 


w  E  Sanders 


THE  LIBRARY 

OF 

THE  UNIVERSITY 
OF  CALIFORNIA 


PRESENTED  BY 

PROF.  CHARLES  A.  KOFOID  AND 
MRS.  PRUDENCE  W.  KOFOID 


THE  METHODS 


OF 


BACTERIOLOGICAL  INVESTIGATION. 


BY 
DE.   FERDINAND   HUEPPE, 

DOCENT  IN  HYGIENE    AND    BACTERIOLOGY    IN    THE    CHEMICAL    LABOEATOBT 
OF  B.  FBESENIUS   AT    WIEBBAIJEN. 


TRANSLATED  BY 

HERMANN"  M.  BIGGS,  M.D., 

INSTBTTCTOB    IN    THE    CAKNEGIB    LABORATORY,   AND  ASSISTANT   TO    THB    OHAIB  OF  PATHO- 
LOGICAL ANATOMY  IN  BBLLEVUB  HOSPITAL  MEDICAL  COLLEGE. 


ILLUSTRATED  BY  THIRTY-ONE  WOOD-CUTS. 


NEW  YORK: 
D.  APPLETON  AND  COMPANY, 

72    FIFTH    AVENUE. 

189T. 


COPfBTGHT,  1886, 

BY  D.  APPLETON  AND  COMPANY. 


DEDICATED  IN  THANKFUL  EE8PEOT 

TO  THE 

GEHEIMEN    KEGIEKUNGSBATH, 
DE.   BOBEET  KOOH. 


AUTHOR'S  PREFACE. 


URGED  by  the  wish  of  my  highly  esteemed  teacher, 
the  Geheimrath  Koch,  I  have  attempted  in  the  fol- 
lowing work  to  meet  the  lack  of  a  comprehensive 
representation  of  the  methods  of  bacteria-investiga- 
tion. It  was  my  endeavor,  as  an  historical  and  ex- 
perimental critic,  to  sift  the  whole  of  the  literature, 
which  was  extraordinarily  scattered  and  in  part  very 
difficult  of  access,  and  to  select  the  good  from  the 
hardly  conceivable  confusion  of  useful  and  useless 
communications,  in  order  to  give  to  the  independent 
investigator  a  useful  hand-book,  and  to  the  beginner 
a  trustworthy  introduction  into  this  territory. 

THE  AUTHOB. 

WIESBADEN,  February,  1885. 


TEANSLATOE'S  PEEFAOE. 


THIS  translation  of  Hueppe's  excellent  work  on 
the  "  Methods  of  Bacteriological  Investigation "  was 
suggested  by  the  want  in  English  of  a  satisfactory 
text-book  on  this  subject  for  the  use  of  students 
working  under  my  direction  in  the  Carnegie  Labo- 
ratory. The  preparation  of  the  original  was  under- 
taken by  Dr.  Hueppe,  at  the  request  of  Prof.  Robert 
Koch,  and  the  work  has  been  thoroughly  and  care- 
fully done.  It  shows  a  complete  familiarity  with 
the  subject  in  hand,  is  comprehensive  in  character, 
and  treats  carefully  all  of  the  approved  methods  of 
investigation. 

Some  difficulties  have  been  met  in  the  transla- 
tion which  those  acquainted  with  this  kind  of 
work  will  readily  understand.  It  is  literal  so  far 
as  is  consistent  with  clearness,  and  no  attempt  has 
been  made  to  attain  elegance  in  style  or  diction ; 
but  it  is  well  known  that  many  German  terms  have 
no  exact  English  equivalent,  and  can  only  be  ren- 
dered accurately  by  a  roundabout  expression.  This 
is  well  illustrated  by  "  Massenkultur "  (as  translat- 
ed by  quantity- culture  on  page  101  et  seq.\  which 
means  a  culture,  whether  pure  or  not,  where  a  great 


6  TRANSLATOR'S  PREFACE. 

quantity  or  bulk  of  bacteria  are  growing;  but, 
since  this  expression  can  not  be  inserted  each  time 
in  the  text,  quantity-culture  will  be  used  wherever 
it  occurs. 

The  labor  of  preparing  the  translation  was  per- 
formed in  the  short  intervals  of  leisure  found  in 
the  midst  of  numerous  other  duties,  and  was  un- 
avoidably interrupted  just  before  its  completion,  so 
that  some  errors  may  be  found  in  the  book,  but  I 
think  that  none  of  serious  importance  have  escaped 
correction. 

The  author  has  requested  me  to  emphasize  the 
fact  uthatx  the  short,  concise  form  chosen  in  the 
work  is  based  on  an  extensive  historical  and  ex- 
perimental review  of  the  whole  subject,  and  that  if 
any  methods,  still  much  used,  have  been  omitted 
or  only  briefly  considered,  it  is  because  they  have 
not  now  the  significance  or  importance  that  has 
been  ascribed  to  them  by  other  writers." 

I  here  desire  to  acknowledge  my  indebtedness 
to  Dr.  L.  W.  Hubbard  and  Dr.  S.  K  Nelson,  both 
of  New  York,  for  valuable  assistance  kindly  ren- 
dered me  in  the  translation. 

The  work  has  been  very  favorably  received  in 
Germany,  and  if  this  translation  meets  only  a 
small  part  of  the  same  consideration  in  America,  I 
shall  feel  well  repaid  for  the  labor  expended. 

HERMANN  M.  BIGGS. 

CARNEGIE  LABORATORY,  NEW  YORK, 
December  1,  1885. 


CONTENTS. 


PACK 

INTRODUCTION      .        .        .        .        .        •       *       *        .        .9 

True  Saprophytic  Forms       .        .        .        .        .        .        .11 

'I.  SPONTANEOUS  GENERATION  AND  THE  PRINCIPLES  OF  STERIL- 
IZATION     .        .        .        .        .        .        •        .        .        .15 

II.  FORMS  OF  BACTERIA  AND  MICROSCOPICAL  TECHNIQUE   .        .    28 

A.  The  True  Endospore  Bacteria 29 

B.  Arthrospore  Bacteria 29 

1.  Arthro-Cocci '     .        .29 

2.  Arthro-Bacteria 29 

8.  Leptothrix 29 

4.  Cladothrix .30 

Determination  of  the  Presence  of  Bacteria  Unstained  .        .    34 

Staining  Bacteria •  .  /  •  .  40 

General  Principles  of  Staining  .  .  >  .  •  .  42 
Preparation  of  Staining  Fluids  .  .  •  •  .48 
Other  Reagents  and  Apparatus  .  .  »  V«:  .61 

Cover- glass  Preparations  .  '  ,  .  .  .  *  .  55 
Examination  for  Tubercle  Bacilli  in  Sputum  ....  .61 
Examination  of  Blood  for  Bacteria  .  .  ,  .  67 

Methods  of  Staining  the  Flagella 73 

Methods  of  Staining  Spores 74 

Preparation  of  Sections         .        .        ,       V       •        •        .78 

III.  CULTURE-METHODS;  PURE  CULTURES    .        .        .        ,        .    92 

1.  Transparent  Fluid  Culture- Media     .        .        «        .  v     .     92 

2.  Fractional  Cultures  .        .        ,   '.   r       .        .  '     .        .  100 

3.  Opaque  Solid  Culture-Media     .        .        .        ...  101 

4.  The  Gelatin-Culture  of  Klebs  and  Brefeld        ...  105 

5.  Method  of  Dilution  .        .   '     .  '     .        .        .       *.       .113 

Method  of  Isolation  by  Heat 119 


8  CONTENTS. 

PAG» 

6.  Cultures  in  Capillary-Tubes,  after  Salomonsen         .        .121 

7.  The  Infection-Methods .125 

8.  The  Cultures  upon  Transparent  Solid  Nutrient  Media, 

according  to  Koch 128 

A.  Transparent  Solid  Media  made  by  the  Addition  of 

Gelatinizing  Substances— " Nutrient  Gelatin"  .  132 

a.  Slide-Cultures 134 

1.  Plate-Cultures  '     .        .        .        .        .        .        .  138 

c.  Test-Tube  Cultures  .  .  .  .  .  .142 

Improvised  Means  .        .        .        ,        .        .        .  146 

B.  Transparent  Solid  Media,  without  the  Addition  of 

Gelatinizing  Substances — Blood-Serum  .        «        .  148 

IV.  INOCULATIONS   FOB  THE   DETEEMINATION   OF  THE   CAUSAL 

KELATION  OF  BACTEBIA-GBOWTH  TO  DECOMPOSITION  AND 
DISEASE 160 

A.  Septic  Bacteria 160 

Anaerobiosis  in  Fluids 164 

B.  Parasitic  Bacteria 172 

Inhalation  Experiments     '    *  •':•-.  *  •  .  .174 

Feeding  Experiments     .  '     .        .  .  .  .  .175 

Cutaneous  Inoculations      .,•'••  .  .  .  .177 

Subcutaneous  Applications    .        .  f  »  .  .178 

Subcutaneous  Injections        .       '.  .  .  .  .179 

Direct  Injection  into  the  Circulation  .  .  .  .  180 

V.  GENERAL  BIOLOGICAL  PEOBLEMS    .     %  .  .  .  .  .183 

Enzyme 185 

Ptomaines     .        .        .        .        .        .        .        .        .        .  187 

Behavior  to  Temperatures 188 

Disinfection  with  Fluids        .        .        .  .        .        .194 

Disinfection  with  Gases 195 

Drying 197 

Action  of  Low  Temperature  and  High  Pressure   .        .        .197 

Electricity .        .        .198 

Phosphorescence 198 

Light 199 

VI.  SPECIAL  HYGIENIC  INVESTIGATION 200 

A.  Earth 200 

B.  Water 202 

a  Air 205 

VII.  BACTERIOLOGY  AS  AN  OBJECT  OF  INSTRUCTION  .  .  210 


BACTERIOLOGICAL   INVESTIGATION. 


INTRODUCTION. 

THE  perfection  of  peculiar  methods  for  the  study 
of  bacteria  was  conditioned,  on  the  one  hand,  by  the 
minuteness  and  rapid  multiplication  of  these  lower 
organisms,  by  reason  of  which  the  formerly  approved 
methods  scarcely  at  all  sufficed  to  determine  their 
morphology  ;  and,  on  the  other,  by  the  biological 
processes  in  which  these  micro-organisms  take  part, 
and  which  the  methods  for  their  study  must  also 
consider. 

In  regard  to  their  biology,  the  bacteria  may  be 
separated  into  two  great  groups :  the  septic  (sapro- 
phytic)  bacteria,  which  feed  on  dead  organic  bodies, 
and  the  parasitic,  which  are  found  in  living  organ- 
isms. 

Among  the  septic  bacteria,  the  true  bacteria  of 
putrefaction  should  be  distinguished  from  those 
which  cause  a  more  typical  decomposition  of  life- 
less organic  matter.  In  the  latter  it  is  possible  to 
even  separate  chemically  the  products  formed.  These 
last  septic  forms  are  designated  as  ferment  bacteria. 
Aside  from  these,  the  pigment  bacteria  also  deserve 
special  notice. 


10  BACTERIOLOGICAL  INVESTIGATION. 

Many  of  these  septic  micro-organisms  need,  tin- 
der all  conditions,  the  oxygen  of  the  air  for  life  and 
activity — the  aerobic  forms  ;  some  species  can  for  the 
time  being  be  deprived  of  this,  and  are  able  even  then 
to  bring  about  their  specific  decomposition,  but  they 
can  also  live  and  multiply  with  a  free  access  of  air. 
These  may  be  designated  as  the  optional  anaerobic 
(facultativ-anaerobiotische)  forms.  Again,  of  other 
forms  it  has  been  asserted  that  the  absence  of  the 
oxygen  of  the  air  is  necessary  for  their  life  and  ac- 
tivity, and  that  they  are  immediately  destroyed  by 
oxygen — the  obligatory  anaerobic  (obligat-anaerobio- 
tische)  forms.  We  know  of  still  other  forms  in  which, 
directly  contrary  to  this,  a  transference  of  the  oxygen 
to  the  medium  in  which  they  grow  takes  place  with 
their  life  activity.  These  cause  an  oxidation  fermen- 
tation. 

Among  the  bacteria  living  as  parasites,  there  are 
forms  which  do  not  complete  their  development  upon 
the  animal  organism,  but  only  occasionally  appear 
as  parasites,  or  pass  through  a  portion  of  their  exist- 
ence in  living  organisms — the  facultative  parasites  of 
van  Tieghem;  other  forms  find,  as  a  rule,  only  in 
living  organisms  all  the  conditions  necessary  for  their 
existence,  but  can  occasionally,  or  in  certain  stages  of 
development,  also  live  as  septic  bacteria — the  facul- 
tative septic  forms  of  de  Bary.  Still  other  forms, 
finally,  seem  to  be  fitted  only  for  the  parasitic  mode 
of  life,  and  appear  quite  incapable  of  passing  any 
period  of  their  existence  as  septic  bacteria.  These 
are  designated  as  the  strongly  obligatory  parasites  of 
de  Bary.  Although  it  is  very  difficult  to  determine 
in  individual  cases  whether  a  parasitic  micro-organism 
belongs  to  this  or  that  group,  yet  in  such  cases  as  the 
recognition  of  the  minute  differences  also  depends 


INTRODUCTION.  H 

more  or  less  upon  the  subjective  opinion  of  the  ob- 
server, it  thus  permits  this  grouping  to  be  regarded  as 
much  freer  from  constraint  as  to  their  real  condition 
than  the  division  of  the  pathogenic  infectious  micro- 
organisms into  endogen  and  ectogen,  which  has  ref- 
erence only  to  the  extremes.  These  last  designations 
permit  only  insufficiently,  either  a  valuation  of  the 
investigations  concerning  the  accessory  causes  of  the 
infectious  diseases,  or  a  strong  emphasis  being  placed 
upon  one  or  another  factor,  and  do  not  allow  the  at- 
tainment of  an  unprejudiced  judgment  upon  matters 
all-important  in  the  aetiology.  There  is  also  the  great- 
est difference  as  to  the  need  of  oxygen  among  the 
parasitic  bacteria.  It  is  to  be  noted,  likewise,  that 
the  parasites  may  be  endophytic — i.  e.,  can  live  in 
the  interior  of  organs  or  cells  ;  perhaps  also  epiphy- 
tic— i.  e.,  can  live  upon  the  surface. 

These  different  phenomena  of  adaptability  can  be 
theoretically  deduced  from  the  simple  septic  mode 
of  life  ;  but  it  is  to  be  remembered  that  direct  inter- 
mediate links  can  seldom  be  determined,  and  that 
individual  forms  can  produce  different  actions. 

TRUE  SAPROPHYTIC  FORMS. 

I.  Ferment  'bacteria.    II.  Pigment  bacteria.    III.  Parasitic  bacteria. 
Aerobic.  Facultative  parasites. 

Forms  produc-       Facultative  anaerobic.       Facultative  septic  forms, 
ing  oxidation  |  | 

fermentation.     Obligatory  anaerobic.  Strongly  obligatory  parasites. 

The  most  general  problem  which  is  presented  in 
bacteria  investigation — viz.,  the  determination  of  the 
group  to  which  a  form  of  bacteria  belongs — can  now 
be  quickly  and  accurately  solved. 

I.  It  is  to  be  determined  whether,  in  decomposi- 


12  BACTERIOLOGICAL  INVESTIGATION. 

tion  or  disease,  bacteria  are  present  or  not.  This 
question  associates  itself  essentially  with  the  question 
of  spontaneous  generation  and  abiogenesis,  teaches 
us  the  value  and  general  principles  of  sterilization, 
and  makes  us  acquainted  with  the  indispensable  re- 
quirements for  reliable  work  in  bacteriology. 

II.  If  bacteria  are  present,  it  is  to  be  determined 
what  forms  they  possess.     This  general  morphologi- 
cal question  demands  special  technical  skill,  as  the 
ordinary  histological  technique  does  not  suffice. 

III.  Each  form  found  to  be  present  is  to  be  culti- 
vated by  itself,  free  from  all  chemical  and  morpho- 
logical admixtures— "pure  cultures."     The  problem 
to  be  solved  by  the  aid  of  pure  cultures  is  a  double 
one — i.  e.,  with  the  help  of  these  the  general  mor- 
phological investigation  is  completed  and  extended, 
and, 

IV.  By  transfers  of  really  pure  cultures  to  decom- 
posable materials  or  susceptible  animals,  it  is  to  be 
determined  whether  the  bacteria  found  are  the  cause 
of  the  decomposition  or  disease. 

By  this  investigation  it  is  certainly  shown  to  which 
of  the  described  groups  a  form  of  bacteria  belongs ; 
then,  extending  likewise  from  the  pure  cultures,  there 
are  yet, 

V.  A  further  series  of  more  exact  biological  prob- 
lems to  be  solved  later,  which,  in  union  with  the  first 
questions,  afford  the  broad  basis  for  theoretical  con- 
sideration and  practical  treatment. 

The  solution  of  all  these  questions  is  to  be  aimed 
at ;  but  experience  has  shown  that  this  is  not  possible 
in  every  case,  and  that  cases  may  come  up  in  which 
one  or  the  other  of  these  cardinal  questions  remains 
unanswered.  For  instance,  as  regards  the  parasitic 
bacteria  which,  in  the  highest  degree,  represent  the 


;     .  INTRODUCTION.  13 

parasitic  adaptability,  viz.,  the  strongly  obligatory 
parasitic  forms,  perhaps  only  the  presence  of  the 
micro-organism  may  be  determined,  while  pure  cult- 
ures can  not  be  obtained.  In  other  forms  of  this 
group,  a  further  step  has  already  been  taken,  by 
which,  to  a  limited  extent,  transfers  to  animals  have 
been  made.  With  individual  pathogenic  bacteria  of 
the  other  groups,  on  the  other  hand,  transfers  are 
not  successful,  because  as  yet  no  species  of  animals 
has  been  shown  to  be  susceptible  to  the  disease  which 
they  produce,  although  pure  cultures  of  these  bac- 
teria have  been  obtained.  (Compare  Methods  of  In- 
fection.) 

In  these  cases  the  greater  attention  should  be  given 
to  those  problems  that  can  be  solved ;  and  it  is  always 
to  be  borne  in  mind  that,  with  a  more  complete  mas- 
tery of  the  methods  in  the  seemingly  most  unprom- 
ising cases,  a  classical  solution  of  all  questions  has 
sometimes  been  obtained. 

The  facts  ascertained  by  means  of  a  perfected 
technique  must  form,  in  every  branch  of  natural  sci- 
ence, the  solid  foundation  upon  which  the  theories 
are  built  up.  No  investigator,  although  in  possession 
of  many  facts,  can  dispense  with  the  guiding  ideas 
that  these  afford ;  but  the  aversion  of  many  investi- 
gators to  every  speculation  is  immediately  aroused, 
for  the  reason  that  by  many  the  deductions  of  natu- 
ral philosophy  are  stated  as  scientific  facts.  Even 
the  subject  of  bacteriology  has  in  this  respect  passed 
through  an  experience  too  sad  to  allow  us  to  forget 
that  the  observations  in  natural  philosophy  can  be 
nothing  else  than  provisional  explanations  of  phe- 
nomena not  yet  really  understood,  the  value  of  which, 
however,  in  suggesting  lines  of  investigation,  is  very 
great.  Eesolutely  must  we  guard  ourselves  against 


14:  BACTERIOLOGICAL  INVESTIGATION. 

placing  under  restraint  such,  deductions,  often  con- 
fused, but  yet  in  harmony  with  facts ;  against  bring- 
ing into  discredit  the  solid  foundation  of  facts  by 
cheap  badinage  of  the  endeavors  of  " learned  ones" 
in  search  of  "little  facts";  or  against  citing  ever 
anew  agreeable  theories  or  refuted  statements  as 
proved  facts,  as  has  often  happened  in  the  domain 
of  bacteriology.  Resolutely  also  must  we  see  to  it 
that  speculations  do  not  form  the  foundation  of  the 
practical  treatment  of  hygiene. 

Against  such  sad  errors  of  speculation  there  is  no 
better  remedy  than  the  familiarity  to  be  obtained 
with  the  methods  (constantly  becoming  more  accu- 
rate) which  are  so  closely  united  to  the  real  advances 
in  the  knowledge  and  possibilities  regarding  the  caus- 
al relation  of  micro-organisms  to  decomposition  and 
disease. 


SPONTANEOUS  GENERATION  AND  THE  PRINCIPLES  OF 
STERILIZATION.* 

SPALLANZANI  f  opened  the  series  of  scientific  ex- 
periments concerning  spontaneous  generation  and  the 
principles  of  sterilization.  He  placed  infusions  of 
organic  substances  in  flasks,  which  were  corked  and 
sealed,  and  then  boiled  for  an  hour  in  a  water-bath. 
These  experiments,  which  form  the  groundwork  of  the 
Appert  method  of  preservation  of  organic  substances, 
can  be  better  made  in  flasks  with  long-drawn-out  necks, 
which  are  closed  during  the  process  of  heating.  Now 
and  then  one  of  these  experiments  (where  the  dura- 
tion of  the  heating  is  only  one  hour)  fails.  But  the 
chief  objection  to  the  conclusions  drawn  from  these 
experiments  lies  in  the  fact  that  no  oxygen  can  gain 
admission  to  the  flask.  In  1810  Gay-Lussac  expressed 
the  opinion  that,  for  the  production  of  fermentation, 
oxygen  must  have  admission. 

The  next  methodical  advance  in  this  direction  con- 
sisted in  the  admission  of  air  into  the  vessel  after 
heating,  this  air  having  been  previously  so  treated 

*  "  Zusainmenfassende  Darstellungen  der  Frage  uber  Abiogenesis ; 
Generatio  spontanea  finden  sich  bei  Gscheidlen."  "  Physiologische 
Methodik,"  Heft  2,  1876,  S.  274;  Heft  4,  1879,  S.  499;  und  bei  Tyn- 
dall,  "  Essays  on  the  Floating  Matter  of  the  Air,"  second  edition,  1883. 

t  "Physikalische  und  mathematische  Abhandlungen,"  1769. 


16  BACTERIOLOGICAL  INVESTIGATION. 

that  it  could  contain  no  germs.  Franz  Schulze  *  de- 
signed a  flask  which  had  a  double  perforated  cork,  in 
which  were  placed  two  glass  tubes  bent  at  a  right 
angle,  and  which  terminated  close  underneath  the 
cork.  The  decomposing  infusions  or  substances  sus- 
pended in  water  were  heated  on  a  sand-bath  until  all 
portions  had  reached  the  temperature  of  boiling  wa- 
ter. Then,  while  the  steam  was  still  escaping,  a  Lie- 
big's  globe  apparatus  was  fastened  to  each  of  the  glass 
tubes.  One  of  the  globes  was  filled  with  concentrated 
sulphuric  acid,  and  the  other  with  a  solution  of  caus- 
tic potash.  Now,  by  the  application  of  suction-force 
on  the  side  to  which  the  apparatus  containing  caustic 
potash  is  attached,  air  is  drawn  into  the  flask  which 
before  its  entrance  must  pass  through  the  sulphuric 
acid.  When  this  is  done,  no  decomposition  takes 
place,  notwithstanding  the  presence  of  the  air ;  but 
it  soon  occurs  if,  after  the  heating,  ordinary  air  is 
allowed  admittance. 

Schwannf  showed  that  "it  is  not  the  oxygen,  or 
at  least  not  alone  the  oxygen,  of  the  atmospheric  air  " 
which  causes  fermentation  and  putrefaction,  by  pass- 
ing the  air  through  mercury  which  was  heated  to  the 
boiling-point.  No  change  occurred  in  the  fluids  after 
this  was  done. 

To  meet  the  objection,  that  by  this  procedure  the 
air  might  be  altered  chemically,  Schroder  and  von 
Duschij:  passed  the  air  through  cotton,  which  was 

*  "  Vorlaufige  Mittheilung  der  Eesultate  einer  experimentellen 
Beobachtung  uber  Generatio  aequivoca."  Poggendorf  s  "  Annalen  der 
Physik,"  1836,  Bd.  39,  S.  48T. 

t  "  Vorlaufige  Mittheilung,  betreffend  Yersuche  fiber  die  "Wein- 
gahrung  nnd  Faulniss."  Poggendorf  s  "Annalen,"  1837,  Bd.  41,  S. 
184. 

J  "  Ueber  Filtration  der  Luft  in  Beziehung  auf  Faulniss  nnd  Gah- 
rung."  "  Annalen  der  Chemie  und  Pharmacie,"  1854,  Bd.  89,  S.  337. 


SPONTANEOUS  GENERATION.  17 

either  placed  in  tubes  connected  with  the  right-angled 
glass  tubes,  or  they  stopped  the  neck  of  the  flask 
with  cotton  during  the  process  of  boiling.  Pasteur  * 
boiled  the  infusions  in  flasks,  the  necks  of  which  were 
long-drawn-out  and  curved  in  different  ways,  with 
only  this  precaution,  that  the  open  end  always  looked 
downward.  In  this  way  the  air  could  enter,  after  the 
heating,  unaltered  and  unfiltered,  and  no  putrefaction 
took  place. 

In  milk,  Pasteur  succeeded  in  preventing  decom- 
position certainly  only  by  elevating  the  temperature 
to  110°  or  112°  C.  in  a  pressure  of  one  and  one  half 
atmospheres.  Schroder  also  showed  the  inefficacy  of 
boiling  to  prevent  decomposition  in  respect  to  single 
substances,  which  he  found  could  be  sterilized  with 
certainty  only  after  a  long-continued  boiling,  or  by 
elevating  the  temperature  in  a  digester  with  a  press- 
ure of  about  two  atmospheres. 

The  digester,  or  steam-kettle,  for  expanded  steam, 
is  used  for  sterilization  with  a  high  temperature. 
The  temperature  required  for  sterilization  ranges  be- 
tween 110°  and  112°  C.,  corresponding  to  a  pressure 
of  from  one  to  two  atmospheres.  Since  an  equaliza- 
tion of  temperature  is  produced  by  the  strong  cur- 
rents in  the  fluid  brought  about  by  the  differences 
in  temperature,  the  best  execution,  reasoning  a  pri- 
ori, ought  to  be  expected  from  this  apparatus,  since, 
in  accordance  with  the  dynamic  theory  of  heat  with 
the  elevation  of  the  temperature,  the  time  required 
for  the  equalization  of  the  temperature,  and  hence 
the  time  for  sterilization,  is  diminished.  Many 

*  "  M6moire  sur  les  corpuscles  organises  qni  existant  dansl'atmos- 
phere."  "  Annales  de  chimie  et  de  physiques,"  III.  Ser.,  T.  64, 1862, 
S.  66.  And  shorter  article,  "  Compt.  rend.,"  Bd.  48, 1 859,  S.  337 ;  and 
ibid.,  Bd.  50,  8.  849. 


18  BACTERIOLOGICAL  INVESTIGATION. 

forms  of  apparatus*  correspond  to  this  theoretical 
postulate,  but  all  f  do  not  satisfy  it  in  all  respects. 
The  further  consideration  is  added  to  this  uncer- 
tainty that  many  substances  are  chemically  altered 
by  the  elevation  of  the  temperature  above  100°  C., 
so  that  the  apparent  advantages  of  the  apparatus, 
as  compared  with  others,  are  in  many  cases  quite 
illusory.  Any  one  who  possesses  a  reliable,  tested 
steam-kettle  (Dampfkessel)  may  safely  use  this, 
especially  if  the  substances  to  be  sterilized  will  bear 
an  elevation  of  the  temperature  above  100°  C. 

If  it  is  desired  to  use  a  temperature  above  100° 
C.,  without  employing  a  steam-kettle,  a  salt-,  oil-,  or 
paraffine-bath  can  be  employed. 

If  boiling  water  is  to  be  used,  then  the  larger 
flasks,  smaller  flasks,  and  test-tubes  are  boiled  di- 
rectly in  the  water-bath,  but  the  water  in  it  should 
have  a  somewhat  higher  temperature  than  the  con- 
tents of  the  glasses.  The  equalization  of  tempera- 
ture in  the  water -bath  is  not  only  attained  cor- 
respondingly slower  than  at  a  lower  temperature, 
but  it  is  so  uncertain  that  it  should  be  practically 
tested. 

If  a  sufficiently  long  boiling  affords  a  real  steril- 
ization, then  the  temperature  of  the  boiling  water 
can  be  better  used  in  the  form  of  streaming  steam. 
The  steam  sterilization-cylinder  of  Koch,  Gaffky,  and 
Loftier  answers  this  purpose  (Fig.  1).  This  consists 
of  a  cylinder  made  of  strong  tin  plate,  about  half  a 
metre  high  and  from  twenty  to  twenty -five  centime- 

*  Fitz,  "  Ueber  Spaltpilzgahrungen."  "  Berichte  der  deutschen 
chemischen  Gesellschaft,"  Bd.  XVII,  1884,  S.  1188. 

t  Koch,  Gaffky,  Loffler,  "  Versuche  uber  die  Verwerthbarkeit 
heisser  Wasserdampfe  zu  Disinfectionszwecken."  "  Mittheilungen 
aus  dem  kaiserlichen  Gesundheitsamte,"  I,  1881,  S.  322. 


SPONTANEOUS  GENERATION. 


19 


tres  in  diameter,  which,  is  surrounded  by  an  asbestos 
covering  to  prevent  the  loss  of  the  heat,  and  is  pro- 
vided with  a  copper  bottom.  In  the 
interior  at  J2,  in  the  lower  third,  is 
placed  a  grate  ;  the  space  under  this 
is  filled  three  fourths  full  of  water, 
which  is  brought  to  the  boiling-point 
by  a  number  of  gas -flames  (three  or 
five  burners)  placed  under  the  vessel. 
It  is  closed  above  by  a  cover  (If) 
made  of  block-tin  covered  with  as- 
bestos, and  is  not  hermetically  sealed, 
so  that  the  steam  can  escape  around 
the  edges.  In  a  hole  in  the  cover  a 
thermometer  (f)  is  placed. 

The  apparatus  may  also  have  somewhat  larger 
dimensions.  But,  if  these  dimensions  are  very  much 
increased,  it  is  necessary  to  use  salt  solution  in  order 
to  keep  the  temperature  of  the  outer  currents  of 
steam  at  100°  C.  If  the  escape  of  the  steam  is  not 
quite  free,  and  the  radiation  of  the  heat  is  prevented, 
the  temperature  of  the  interior  of  the  cylinder  in  this 
way  may  be  kept  equal  throughout ;  and  since  the 
cover  is  not  hermetically  sealed,  the  temperature  of 
the  steam  does  not  exceed  the  boiling-point,  but 
gives  the  temperature  of  boiling  water,  correspond- 
ing to  the  conditions  of  pressure — that  is,  with  the 
barometer  at  nearly  its  normal  height,  about  100°  C. 

The  advantages  of  this  apparatus,  in  comparison 
with  the  steam-kettle,  are  its  cheapness  and  the  im- 
possibility of  exceeding  the  temperature  of  100°  C. 
when  water  is  used,  so  that  all  substances  can  be 
sterilized  with  this,  which  will  bear  a  temperature  of 
100°  C.  The  equalization  of  the  temperature  is  very 
soon  reached,  and  does  not  undergo  such  oscillations 


20  BACTERIOLOGICAL  INVESTIGATION. 

as  with  the  steam-kettle,  because  the  technical  use  of 
the  apparatus  can  scarcely  be  simpler,  and  on  this 
account  this  element  is  reduced  to  the  minimum. 
The  currents  of  steam  are  far  superior  to  the  water- 
bath,  through  the  certainty  of  their  action  and  the 
relatively  short  time  required. 

These  practical  advantages  render  this  apparatus 
the  most  desirable  one  for  all  cases  in  which  high 
temperatures  are  used  for  sterilization,  in  spite  of  the 
fact  that,  theoretically,  to  compensate  for  the  some- 
what lower  temperature,  it  must  be  used  longer  than 
would  be  necessary  for  expanded  steam  at  a  tempera- 
ture above  100°  C.  The  beginning  of  the  heating 
not  being  included,  the  time  required  for  the  sterili- 
zation varies  from  one  half  to  two  hours,  depending 
on  the  size  of  the  object. 

A  vessel  which  fits  in  the  cylinder  is  included 
with  the  apparatus.  In  this  are  placed  the  small 
flasks  or  tubes  to  be  sterilized,  and  to  the  handle  of 
the  vessel  or  to  the  neck  of  the  larger  flasks  a  string  is 
fastened  for  conveniently  raising  and  lowering  them. 
This  string  is  made  fast  to  the  hook  k,  to  be  found 
on  the  rim  of  the  cylinder. 

But  many  substances  are  altered  by  exposure  to  a 
temperature  of  100°  C.,  and  especially  coagulation  of 
the  albumen  is  always  brought  about.  In  order  to 
avoid  this,  the  discontinuous  sterilization  of  Tyndall 
is  used.  The  principle  of  "  Sterilization  by  Discon- 
tinuous Heating "  (I.  c.,  pp.  210  and  337)  was  founded 
on  the  observation  that  living  bacteria  are  killed  by 
exposure  to  a  relatively  low  temperature,  below  the 
point  required  for  the  coagulation  of  albumen,  while 
the  spores  are  not  destroyed  by  these  low  tempera- 
tures,* but  are  easily  killed  after  germination.  If 

*  Oohn,  "  Untersuchungen  fiber  Bacterien,"  IV  ;  "Die  Bacterien 


SPONTANEOUS  GENERATION.  21 

the  fluid  to  be  sterilized  is  exposed  for  one  or  two 
hours  to  a  temperature  of  from  52°  C.  to  65°  C.,  only 
the  living  bacteria  are  in  this  way  destroyed,  and  per- 
haps not  even  all  of  these  the  first  time.  The  resistant 
spores  possibly  present  in  the  solution  thus  treated 
germinate  some  on  the  first  and  second,  others  on  the 
third  and  following  days.  If  now  the  fluid  is  exposed 
to  the  same  temperature  as  before  on  the  second  and 
third  days,  the  living  bacteria,  or  those  that  have  de- 
veloped from  the  spores,  are  killed  each  time,  so  that, 
if  this  operation  is  continued  long  enough,  it  is  pos- 
sible to  sterilize  with  certainty  all  fluids  below  the 
temperature  producing  chem-  Fio  2 

ieal  alterations.  In  general, 
exposure  to  a  temperature  of 
about  58°  C.  for  one  or  two 
hours,  on  from  five  to  eight 
successive  days,  is  recommend- 
ed. This  may  be  done  by  the 
use  of  the  water-bath,  but  more 
conveniently  with  the  appara- 
tus shown  in  Fig.  2.  This  con- 
sists of  a  double-walled  cyl- 
inder made  of  copper.  The 
chamber  between  the  two  walls 
is  about  half  filled  with  water,  and  the  cylinder  itself 
well  closed  with  a  double- walled  cover,  which  is  also 
filled  with  water.  The  cover  has  upon  its  side  a  hol- 
low tube  (d\  whose  lumen  communicates  with  the 
chamber  in  the  cover.  This  is  warmed  by  the  flame 
(dl\  while  the  cylinder  itself  is  warmed  from  below 
by  a  flame  placed  under  it.  There  are  three  tubes  in 
the  cover,  one  of  which  (c)  is  used  for  filling  the  cover 

und  die  Urzeugung."  "  Beitrage  zur  Biologie  der  Pflanzen,"  Bd.  II, 
Heft  2,  S.  249,  1876. 


22  BACTERIOLOGICAL  INVESTIGATION. 

and  for  receiving  a  thermometer  which  indicates  the 
temperature  of  the  water  in  it ;  a  second  (bl)  receives 
a  thermometer  which  passes  into  the  air-chamber  (5) 
of  the  cylinder ;  and  the  middle  tube  (a1)  receives  a 
thermometer  which  passes  into  a  small  central  cylin- 
der (a),  the  cavity  of  which  communicates  with  the 
water-chamber  on  the  exterior.  The  outer  water- 
chamber  is  filled  by  means  of  a  tube  placed  on  the 
side. 

The  principle  of  discontinuous  sterilization  may 
also  be  used  in  many  cases  as  discontinuous  boiling, 
since  there  are  many  substances  which  are  altered  by 
long  boiling  that  are  not  materially  changed  by  brief 
but  often  repeated  boiling.  If  such  substances — as, 
for  example,  gelatin — are  boiled  for  a  short  time  on 
four  or  five  successive  days,  it  is  possible  to  sterilize 
them  with  certainty. 

For  refuting  other  objections  which  are  made  as 
to  the  existence  of  spontaneous  generation,  it  znay  be 
desirable  to  use  substances  which  have  not  even  once 
been  subjected  to  the  lower  temperatures— i.  e.,  tem- 
peratures below  the  coagulation-point  of  albumen. 

This  object  is  attained  in  two  ways :  first,  by  free- 
ing the  solution  by  filtration  from  possible  admixt- 
ures ;  or,  secondly,  by  endeavoring  to  obtain  the 
same  uncontaminated  from  the  beginning. 

In  respect  to  the  first,  Helmholz*  observed  that 
the  fermentation  produced  by  yeast  did  not  pass 
through  a  membrane ;  on  the  contrary,  this  did  occur 
with  putrefaction.  Consequently  such  membranes  are 
not  available  for  this  purpose.  Positive  results  were 
first  obtained  by  Tiegel,f  who  succeeded  in  separating 

*  "  Ueber  das  Wesen  der  Faulniss  und  Gahrung."  Muller's 
"  Archiv  fGr  Anatomie  und  Physiologie,"  1843,  S.  453. 

f  "  Correspondenzblatt  fur  schweizer  Aerzte,"  1871,  S.  275 ;  und 


SPONTANEOUS  GENERATION.  23 

mechanically — by  filtration  of  septic  fluids  through 
clay  cells,  using  positive  or  negative  pressure  on 
one  side — the  septic  material  from  the  quite  inactive 
fluid.  Miquel  and  Benoist  *  endeavored  to  remove  the 
germs  by  a  gypsum  filter.  They  took  glass  balloons 
with  drawn-out  necks,  in  the  narrow  portion  of  which 
an  asbestos-stopper  was  placed,  and  beyond  this  a 
layer  of  gypsum.  Before  use  the  apparatus  is  slowly 
heated  to  170°  C.,  then  the  fluid  to  be  filtered  is  slow- 
ly poured  upon  the  gypsum-stopper.  The  stopper 
consists  of  1-6  asbestos,  52*1  gypsum,  46  water.  The 
diluted  juice  of  flesh  and  plants  and  urine  filtered 
rapidly,  serum  and  albuminous  fluids  somewhat  more 
slowly ;  all  were  germ  free  and  free  from  life.  A  cap- 
illary tube,  cemented  underneath  the  constriction, 
by  being  united  to  an  aspirator,  permits  the  reduc- 
tion of  the  pressure  in  the  balloon. 

Gautier  f  used  a  very  long-necked  flask  of  faience 
or  unglazed  porcelain,  which  tapered  below  into  a 
cone.  Through  this  porous  cone,  the  real  filter,  the 
fluid  to  be  filtered  passed  from  without  into  the  inte- 
rior of  the  porcelain  flask.  For  rarefying  the  air  in 
the  neck  of  the  flask,  a  glass  tube,  bent  at  a  right  an- 
gle, is  fastened  by  vermilion-paint  cement,  so  that 
the  shank  reaches  down  to  the  bottom  of  the  cone 
while  the  other  outer  end  tapers  into  a  small  cone 
and  exactly  fits  into  a  corresponding  conical  expan- 
sion of  a  second  tube.  This  second  glass  tube  is  like- 
wise bent  at  a  right  angle,  and  the  end,  which  is 

"  TJeber  die  fiebererregende  Eigenschaft  des  Mikrosporen  septicum." 
Dissert.  Bern,  1871.  Citirt  nach  Klebs. 

*  "  Bulletin  de  la  societS  chimique  de  Paris,"  1881,  Bd.  35,  S. 
552. 

t  "Sterilization  &  froid  des  liquides  fermentescibles."  "Bulletin 
de  la  societe  chimique,"  1884,  Bd.  42,  S.  146. 


24  BACTERIOLOGICAL  INVESTIGATION. 

united  with  the  portion  of  the  first  tube,  possesses  a 
conical  expansion,  while  the  other  encf  reaches  to  the 
bottom  of  a  glass  flask  with  a  narrow  neck.  To  the 
side  of  this  glass  flask  a  tube  with  a  conical  expan- 
sion is  cemented.  The  two  conical  expansions  are 
closed  with  cotton,  and  then  this  glass  balloon  with 
its  projecting  portion  and  the  glass  tubes  are  steril- 
ized. In  the  same  way  the  porcelain  flask  with  its 
glass  tube  is  heated,  and,  after  removal  of  the  cotton 
stopper  from  the  cone  of  the  first  glass  tube,  this  is 
inserted  into  the  conical  expansion  of  the  second.  In 
the  conical  expansion  of  the  projection  from  the  glass 
flask,  after  the  removal  of  the  stopper,  a  glass  tube, 
extending  out  into  a  corresponding  cone,  is  intro- 
duced, which  is  filled  with  heated  asbestos.  The 
joints  uniting  the  conical  expansions  with  the  corre- 
sponding conical  constrictions  are  covered  with  shel- 
lac. By  aspiration  at  the  free  end  of  the  asbestos- 
tube,  the  air  in  the  entire  apparatus  is  rarefied,  and, 
when  the  cone  of  the  porcelain  flask  is  immersed  in  a 
fluid,  by  the  existing  negative  pressure,  fluid  which 
is  quite  free  from  germs  is  aspirated  into  the  porce- 
lain flask.  The  vermilion-paint  cement,  prepared 
with  oil  of  turpentine,  consists  of 

Boracic  acid  (crystallized) 8  parts. 

Silicic  acid 2     " 

Vermilion 12     " 

Most  of  the  filtrates  obtained  free  from  germs  in 
this  way  have  experienced  no  material  alterations ; 
but  some  of  them  do  not  remain  unchanged  after 
the  filtration,  as  the  filter  does  not  allow  all  materials 
to  pass  through  equally  well,  and  albuminous  solu- 
tions are  often  quite  seriously  altered  quantitatively 
and  qualitatively. 

In  order,  also,  to  eliminate  this  possible  error,  the 


SPONTANEOUS  GENERATION.  25 

substances  should  be  obtained,  quite  uncontaminated, 
by  the  use  of  the  greatest  cleanliness.  Before  each 
manipulation  the  hands  are  cleansed  with  a  one  per 
cent  solution  of  corrosive  sublimate,  and  are  then 
rinsed  in  sterilized  water,  or  the  sublimate  is  removed 
by  alcohol,  the  alcohol  by  ether,  and  the  latter  al- 
lowed to  evaporate.  All  vessels  are  well  sterilized  ; 
the  manipulations  and  all  operations  are  performed 
very  rapidly  and  with  the  antiseptic  precautions  of 
modern  surgery.  In  this  manner  it  is  possible  to  ob- 
tain blood,  milk,  urine,  etc.,  without  anything  ever 
being  mingled  with  the  same,  or  without  decompo- 
sition taking  place.  Some  details  follow  later  in  the 
methods  of  infection  and  in  the  inoculation  experi- 
ments. No  organism  is  ever  formed,  not  even  a  mi- 
crococcus,  either  from  the  unorganized  material,  or 
from  " molecules  of  nitrogen,"  or  from  microscopic 
forms  of  life,  or  from  the  anamorphosis  of  proto- 
plasm, which  fact  certainly  has  not  prevented  many 
authors  from  mistaking,  for  real  cocci,  bodies  of  dif- 
ferent origin  showing  molecular  motion,  from  which 
also  later,  bacilli,  etc.,  should  develop.  Whoever  has 
obtained  contrary  results,  upon  the  ground  of  a  few 
experiments,  must  first  show  that  he  has  mastered 
the  technique^  as  was  the  case  in  the  many  positive 
results  of  van  den  Broek,  Pasteur,  Roberts,  Lister, 
Cheyne,  and  Meissner.  The  technical  dexterity  nec- 
essary for  this  is  only  to  be  obtained  by  many  indi- 
vidual experiments. 

By  these  experiments,  arranged  for  spontaneous 
generation  (which,  especially  through  Pasteur,  have 
taken  a  form  easy  of  mastery),  and  by  the  disinfection 
experiments  of  Koch,  the  still  remaining  principles 
of  sterilization  have  been  reduced  to  a  reliable  and 
convenient  form. 


26 


BACTERIOLOGICAL  INVESTIGATION. 


Metal  objects — scissors,  knives,  pincettes,  plati- 
num-needles— are  first  mechanically  cleaned,  then 
are  heated  in  the  flame,  and  for  cooling  are  laid  upon 
a  sterilized  glass  plate  and  protected  from  dust  by  a 
bell-jar. 

Glass  objects — glass  plates,  slides,  flasks,  test-tubes 
— are  first  mechanically  cleaned,  then,  if  they  are  very 
dirty,  greasy,  or  have  been  used  for  other  purposes, 
are  dipped  in  concentrated  sulphuric  acid  or  hydro- 
chloric acid,  and  finally  repeatedly  washed  in  dis- 
tilled water  until  every  particle  of  the  acid  has  been 
removed.  The  water  is  first  allowed  to  run  off,  and 
the  articles  thoroughly  dried  in  the  dry-oven,  or  it 
is  removed  by  means  of  alcohol,  in  the  following 
manner :  they  are  dipped  into  alcohol,  and  the  last 
particles  of  this  are  removed  by  ether,  which  is  al- 
lowed to  evaporate.  According  to  the  degree  of  un- 
cleanliness  of  the  vessels  in  the  beginning,  either  this 
whole  procedure  must  be  gone  through  with,  with 
the  greatest  care,  or  it  is  sufficient  to  wash  them  in 
distilled  water.  The  chemically 
clean  vessels  must  then  be  freed 
from  germs.  For  this  purpose 
the  vessels,  having  their  necks 
stopped  with  a  closely  fitting 
mass  of  cotton,  are  immediately 
placed  in  the  double- walled  dry- 
oven  (Fig.  3).  This  is  provided 
with  a  thermo-regulator  (r)  and 
a  thermometer  (t).  The  test- 
tubes  are  more  conveniently 
placed  in  a  basket  (d)  made  of 
wire,  which  will  hold  a  large 
number.  Catheters,  syringes, 
pipettes,  capillary- tubes,  and  other  glass  tubes  are 


SPONTANEOUS  GENERATION.  2T 

placed  in  a  clean  glass  and  then  put  in  the  dry- oven. 
All  such  glass  objects  should  remain  at  least  two 
hours*  at  a  temperature  of  150°  or  160°  C.,  the  time 
required  to  raise  the  temperature  to  this  point  not 
being  included.  The  objects  are  allowed  to  cool  in 
the  oven,  so  that  they  can  be  removed  directly  before 
use,  or  at  least  care  should  be  taken  to  protect  them 

from  dust. 

Cork  stoppers  are  to  be  avoided.  Rubber  corks, 
caps,  and  bands,  etc.,  are  sterilized  in  the  steam- 
cylinder  for  from  three  quarters  of  an  hour  to  one 
hour. 

Most  vessels  with  their  contents  should  be  steril- 
ized once  more ;  after  which  it  is  advantageous  to 
bind  over  the  tops  two  layers  of  filter-paper,  since  the 
cotton  stoppers  are  proof  against  bacteria,  but  not  al- 
ways against  fungi. 

Previous  experience  has  shown  that  germs  from 
the  air  are  more  seldom  the  cause  of  failure  than 
the  unintentional  infection  through  unclean  or  insuf- 
ficiently sterilized  vessels,  and  the  manipulation  with 
hand  and  instruments  not  certainly  sterilized. 

*  Practically  I  have  found  that  exposure  to  a  temperature  of  150° 
or  160°  0.  for  from  fifteen  to  twenty  minutes  answers  exactly  the 
same  purpose,  and  it  has  been  repeatedly  shown  that  neither  germs 
nor  their  spores  will  withstand  exposure  to  so  high  a  temperature 
for  this  length  of  time. — TK. 


II. 

FORMS  OF  BACTERIA  AND  MICROSCOPICAL  TECH- 
NIQUE. 

IN  the  examination,  under  the  microscope,  of  a 
substance  containing  bacteria,  different  forms  present 
themselves,  whose  general  morphological  peculiari- 
ties, on  the  one  hand,  and  whose  differences  or  like- 
nesses, on  the  other,  must  be  determined. 

The  forms  are  determined  by  the  use  of  the  gen- 
eral microscopical  technique,  in  its  peculiar  applica- 
tion to  bacteria,  while  the  second  question  can  only 
be  solved  after  obtaining  pure  cultures. 

The  forms  of  bacteria  (Fig.  4)  are  round  (1,  3,  4, 


P  0        O  0      CO 
O         Q0       CO 


.22 


'!fc      iff      N^ 

•H          |  /  §        ^ 
/  tf 


^Ox    * 


^»  29       ~      o 

nnnn^  ./     «  xo  **r/l    / 

^  °^  000 


oOOD/b  r40f 

In  part  from  Koch  and  Prazmowski. 


BACTERIA  AND  MICROSCOPICAL  TECHNIQUE.     29 

and  5),  oval  (2),  shorter  (6)  or  longer  (7)  rod-form  cells  ; 
and,  in  addition,  curved  rods  (11,  12)  and  spiral-formed 
organisms  (13-16)  are  observed.  These  forms  appear 
sometimes  isolated,  sometimes  united  in  a  definite 
manner  (3,  4,  5). 

The  next  object  to  be  attained  is  the  separation  of 
these  forms  and  the  grouping  of  them  as  naturally  as 
possible,  so  that  a  general  examination  of  the  mor- 
phology of  the  bacteria  may  be  made. 

A.  THE  TEUE  ENDOSPOEE  BACTERIA  (Fig.  4).— 
These  multiply  sometimes  by  division,  sometimes 
also  by  the  endogenous  formation  of  spores. 

1.  Cocci.  -|  a>  Eound-  I  CeUs.  1  Tendency  to  the  for- 

**-*iL  1     inationofzooglcea. 


2.  Straight  rods,    C  £  '  Tendency  to 

(c.  Clostridium-forms.  mat  ion    of    fila- 

{      ments,  desmo-bac- 

3.  Curved  rods,  vibriones.  f     teria  ;  the  filaments 

show  no  variation 
from  end  to  end. 

4.  True  spirilla-forms,  spirilla,  and  spirochaetas. 

B.  AETHEOSPOEE  BACTERIA.  —  These  forms  also 
multiply  by  division,  bat  they  do  not  produce  endoge- 
nous spores.  On  the  other  hand,  single  individuals 
may  separate  themselves  from  the  colonies  and  form 
new  generations.  These  individuals  appear  almost 
constantly  as  round  cells,  gonidia,  or  arthrospores. 

1.  ArtTiro-CoccL  —  These  consist  only  of  cocci,  and, 
by  the  union  of  the  cocci,  form  torula. 

2.  ArtTiro-Bacteria.  —  These  form  round  cells  simi- 
lar to  cocci,  but  also  short  rods,  long  rods,  and  fila- 
ments, which  show  no  variation  from  end  to  end. 

3.  Leptothrix.  —  These  form  cells  similar  to  cocci, 
rods,  spirals,  and  filaments,  which  show  a  variation 
from  end  to  end. 


30 


BA  GTE  BIO  L  0  GIGAL  INVESTIGA  TION. 


FIG.  5. 


4.  CladotTirix  (Fig.  5). — These  form  cocci,  rods, 

filaments,  and  spi- 
rilla. The  fila- 
ments form  false 
ramifications  (A). 

The  arthro- coc- 
ci and  arthro-bac- 
teria  have  as  yet 
been  very  little 
studied,  and  are 
perhaps  to  be 
classed  with  the 
true  bacteria. 
These  two  groups, 
so  far  as  they  have 
been  studied,  seem 
to  stand  nearer  to 
the  fission  algse  on 
account  of  their 
form.  Zopf  *  class- 
es them  directly 
with  the  bacteria, 
because  of  the  ap- 
pearance of  forms 
similar  to  these  in 
their  development. 

Cladothrixdiehotoma;  after  Zopf.    A,  branching  ^ne   nex*   (lnes- 

plant  with  slightly    and    decidedly    spiral  tion    in    the 
branches.     B^  spiril,  one  end  of  which  is 

more  winding  than  the  other.     Z>,  branch  SCOplCal 
with  narrow  and  broad  windings.     E,  spir-          , .         . 

ils ;  a,  undivided,  6,  divided  into  rods,  and  gatlOU  IS    tO 

at  c  into  Zopf  s  cocci.    F,  spirocheeta  form ;  mf-rip  wT|i/,}i    of    fhp 

at  a,  undivided,  at  6,  schematic  division  into  mine  WniCU   C 

long  rods,  at  c,  into  short  rods,  and  at  d  in  cocci,  described          f  OrmS 

are,  in  general,  present.     After  obtaining  a  pure  cult- 
*  Compare  ray  criticism  of  the  work  of  Zopf  in  the  "Fortschrit- 
ten  der  Medizin,"  1883,  No.  6. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     31 

ure,  it  must  then  be  determined  what  is  the  typi- 
cal form ;  whether  it  remains  the  same  under  all 
conditions,  so  that  it  not  only  has  the  value  of 
growth-form,  but  also  constitues  a  form-species  or 
form-genus ;  or  whether  it  corresponds  to  external 
conditions,  being  sometimes  larger  and  broader,  some- 
times smaller  and  thinner ;  or  whether,  finally,  other 
forms  may  appear  in  their  development.  Further, 
it  ought  to  be  ascertained  whether,  in  the  develop- 
ment of  a  species,  a  single  form  or  a  form- cycle  ap- 
pears or  can  appear. 

In  regard  to  the  individual  peculiarities,  there  re- 
main yet  the  following  points  to  be  noted,  although 
those  already  referred  to  seem  to  be  numerous.  In 
the  spiral  forms,  Fig.  4  (13,  14,  15,  16),  the  number 
and  the  arrangements  of  the  typical  spirils  should 
be  observed  ;  then  it  is  to  be  noted  whether,  in  divis- 
ion, the  spirils  separate  into  curved  rods  similar 
to  the  vibriones,  and  whether  these  fissions-products 
again  develop  into  typical  spirils,  or  whether  the 
division  extends  on  to  the  production  of  forms 
which  are  similar  to  the  rod-bacteria  and  cocci, 
which  finally  originate  new  generations.  If  re- 
agents are  used,  it  is  first  necessary  to  note  the 
stage  of  development,  since,  either  during  the  pro- 
cess of  division  or  immediately  before  it,  the  re- 
agents will  make  visible  the  lines  of  division  in  place 
of  the  apparently  homogeneous  character  of  the  fila- 
ments. On  the  other  hand,  the  value  of  such  chem- 
ical action  should  not  be  overestimated,  since,  in  the 
height  of  their  development,  that  most  susceptible 
reagent,  photography,  shows  not  the  slightest  trace 
of  union. 

In  the  case  of  the  vibriones,  Fig.  4  (11,  12)— the 
smallest  forms  of  which  (11)  Koch,  on  account  of 


32  BACTERIOLOGICAL  INVESTIGATION. 

their  characteristic  appearance  with  the  usual  mag- 
nifying power,  called  comma-bacilli — it  should  be 
noticed  whether  they  divide  into  smaller  vibriones, 
or  whether  they  can  form  spherical  and  rod-shaped 
forms.  Through  the  union  of  a  number  of  vibriones, 
S-forms  and  long,  slender  filaments  similar  to  spirilla 
(11)  may  be  produced  which  the  inexperienced  ob- 
server can  easily  confound  with  the  true  spirilla  (13). 
These  spiral  filaments,  according  to  the  degree  of 
curvature  of  the  individual  vibriones,  form  sometimes 
slightly  wavy  lines,  sometimes  spirils  with  narrow 
windings.  The  spiral  forms  of  the  vibriones  are  to 
be  considered  as  a  kind  of  thread-formation,  which, 
as  contrasted  with  the  true  spirilla,  appear  only  un- 
der certain  conditions,  as,  for  example,  the  partial 
or  total  exhaustion  of  the  culture-medium,  and  never 
show  the  regularity  of  the  latter. 

In  the  rod- formed  bacteria  it  should  be  observed 
whether  the  division  of  long  rods  into  short  ones,  or 
into  oval  and  round  cells,  predominates.  Some  rod- 
forms,  by  their  union,  produce  long  filaments,  while 
others  show  a  tendency  to  the  formation  of  zooglcea. 
On  account  of  the  greater  tendency  of  the  short-rod 
bacteria,  like  the  cocci,  to  form  zoogloea,  Cohn  sepa- 
rated them  from  the  vibriones  and  long-rod  bacteria, 
which  he  classifies  as  filament-bacteria.  It  is  to  be 
noted  whether  the  character  of  the  culture-medium 
(solid  or  fluid)  has  any  influence  upon  this. 

In  the  cocci  it  should  be  determined  whether  the 
oval  cocci  produce,  by  their  division,  smaller  oval 
cells  or  round  cells,  and  whether  the  round  cocci,  be- 
fore then*  division,  become  oval,  and,  finally,  whether 
the  oval  cocci  become  short  rods  previous  to  their 
division. 

The  appearance  of  straight  rods  may  be  produced 


BACTERIA   AND  MICROSCOPICAL   TECHNIQUE.     33 

in  the  curved  rods,  when  the  concavity  or  convexity 
of  the  rod  is  turned  upward.  A  straight  rod  stand- 
ing upright  may  present  the  appearance  of  a  round 
cell,  and  a  long  rod  placed  at  an  angle  to  the  plane 
of  vision  may  appear  like  a  short  rod.  It  is  only 
possible  to  decide  such  questions  after  many  single 
observations  and  with  the  use  of  pure  cultures. 
Many  hundred  observations  of  mixtures  of  bacteria 
do  not  serve  at  all  for  the  decision  of  morphological 
questions. 

The  consideration  of  spore-formation,  Fig.  4  (5  Z>,  8, 
9, 10,  12),  and  germination  shows  two  different  types. 
The  rods  may  develop  into  filaments  before  the  forma- 
tion of  spores  (8),  or  the  spores  may  appear  in  the 
mobile  rods  (9)  ;  sometimes  they  are  formed  in  the 
interior  (8,  9),  sometimes  near  the  end  (12).  The  rods 
often  become  whetstone-  or  club-shaped  before  the 
formation  of  the  spores  (clostridium,  10).  Under 
other  conditions,  quite  abnormal,  so-called  involution 
forms  are  observed  (17). 

The  work  of  Cohn  *  is  fundamental  as  regards  the 
value  of  form.  In  this  he  first  clearly  described  the 
relations  of  the  growth-form,  the  form-genus,  form- 
species,  true  varieties,  form- varieties,  and  physiologi- 
cal varieties.  This  work  is  not  only  often  entirely 
misunderstood  by  his  opponents,  but  also  construed 
in  an  opposite  sense  by  his  own  zealous  supporters, 
who  have  repeatedly  interpreted  his  arguments  for 
the  existence  of  true  species  among  the  bacteria 
in  the  sense  of  a  belief  in  constancy  in  species 
and  form,t  which  was  accepted  before  the  Darwinian 
theory. 

*  "  Untersuchungen  liber  Bakterien."  "  Beitrage  ziir  Biologie 
der  Pflanzen,"  Bd.  I,  2.  Heft,  S.  127,  1872.  2.  Abdruck,  1881. 

t  For  the  further  investigation  of  these  questions,  consult  Naegeli, 


34:  BACTERIOLOGICAL  INVESTIGATION. 


DETERMINATION   OF  THE  PRESENCE   OF  BACTEEIA 
UNSTAINED. 

The  oldest  method  of  examining  unstained  bac- 
teria consisted  in  mingling  a  small  drop  of  fluid  or  a 
particle  of  matter  containing  bacteria,  with,  a  drop  of 
indifferent  fluid,  placing  it  upon  a  slide,  putting 
over  it  a  cover-glass,  and  then  examining  it  accord- 
ing to  the  method  of  histological  procedure. 

The  picture  presented  in  these  cases  is  of  the  same 
nature  as  in  unstained  tissue-preparations,  especially 
in  this :  that  the  objects,  because  of  their  different 
power  of  refracting  light  from  the  inclosing  media,  or, 
according  to  Koch,  "on  account  of  the  refraction  of 
the  rays  of  light  passing  through,  present  a  picture 
of  lines  and  shadows— viz.,  the  structure-picture." 

In  these  cases  a  diaphragm  is  used,  as  in  other 
histological  work  in  which  it  is  desired  to  make  out 
the  structure-picture.*  The  diffused  daylight  will 

"Die  niederen  Pilze  in  ihrenBeziehungen  zu  den  Infectionskrankheiten 
und  der  Gesundheitspflege,"  1877.  Naegeli  und  Buchner,  in  Naegeli'8 
"  Untersuchungen  iiber  niedere  Pilze,"  1882.  Koch,  "  Zur  Aetiologie 
desMilzbrandes,"  "  Mittheilungen  aus  dem  kaiserlichen  Gesundheits- 
amte,"  Bd.  I,  1881,  S.  49.  Gaffky,  "  Experiraentell  erzeugte  Septi- 
caeraie  mit  Rucksicht  anf  progressive  Virulenz  und  accommodative 
Zuchtung,"  ibid.,  S.  80.  Flugge,  "  Fermente  und  Mikroparasiten," 

1883,  und  "  Deutsche  raed.  Wochenschrift,"  1884,  No.  46.   Zopf,  "  Die 
Spaltpilze,"  2.  Aufl.,  1884.     De  Bary,  "  Vergleichende  Morphologic 
und  Biologie  der  Pilze,"  1884.     Huppe,  "  Fortschritte  der  Medi- 
zin,"  1883,  No.  6,  und  1884,  No.  6  (Kritik  der  Ansichten  von  Zopf), 
und  "  Ueber  die  Zersetzungen  der  Milch  und  die  biologischen  Grund- 
lagen  der  Gahrungsphysiologie,"  "Deutsche  med.  Wochenschrift," 

1884,  Nos.  48-50. 

*  Zur  weiteren  Orientirung  fiber  das  Mikroskop  und  die  mikro- 
skopische  Technik:  Dippel,  k'Das  Mikroskop,"  2.  Aufl.,  I,  1882-83; 
Frey,  "  The  Microscope  and  the  Microscopical  Technique  " ;  Strass- 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     35 

not  suffice  to  illuminate  the  object  when  the  higher 
powers  are  used,  so  that  it  is  necessary  to  employ  a 
condenser,  which  does  not  define  the  structure-pict- 
ure, but  serves  to  increase  the  illumination.  For  this 
kind  of  microscopical  work  the  achromatic  condens- 
ers of  the  larger  English  instruments  answer  the  pur- 
pose better  than  any  others. 

The  dry  system  of  objectives  does  not  suffice  for 
most  bacteria ;  in  order  to  make  out  only  approxi- 
mately the  correct  forms,  one  must  use  in  this  inves- 
tigation the  immersion  system,  in  which,  according 
to  Amici,  the  stratum  of  light,  by  passing  through  a 
stronger  refracting  medium,  compensates  as  much  as 
possible  for  the  error  caused  by  the  dispersion  of  the 
rays  by  the  cover-glass. 

Since  the  cover-glass  and  the  front  lens  of  the  ob- 
jective consist  of  crown-glass,  and  water,  on  account 
of  its  low  refractive  power,  does  not  quite  correct  the 
error,  in  the  water-immersion  systems  correction-ad- 
justers and  cover-glasses  of  a  certain  thickness  are 
necessary. 

But  the  correction  is  obtained  if  the  immersion- 
fluid  has  the  same  exponent  of  refraction  as  crown- 
glass.  Such  a  fluid,  according  to  Abbe,*  presents  an 
optical  homogeneous  union  between  the  preparation 
and  the  objective,  which  prevents  all  refraction  of  the 
rays  in  front  of  the  convex  surface  of  the  optical  sys- 
tem. By  this  means  the  loss  of  light  through  reflec- 
tion at  the  natural  joints  of  the  different  optical 
media  is  avoided,  and  at  the  same  time  a  very  con- 
burger,  "  Das  botanische  Practicum,"  1884 ;  Friedlander,  "  Micro- 
scopical Technique." 

*  "  Ueber  Stephenson's  System  der  homogenen  Immersion. "  "  Sitz- 
ungsberichte  der  Jenaischen  Gesellschaft  f.  Med.  und  Naturw.,"  1879, 
10.  Januar. 


36  BACTERIOLOGICAL  INVESTIGATION. 

siderable  amount  of  spherical  aberration  is  prevented. 
On  account  of  this,  the  correction-adjusters  necessary 
in  the  water-immersion  systems  can  be  dispensed 
with,  and  the  thickness  of  the  cover-glass  is  of  no 
great  moment,  since,  as  soon  as  the  intervening  medi- 
um has  the  same  index  of  refraction  and  dispersion 
as  the  cover-glass,  the  same  result  for  optical  pur- 
poses is  obtained  whether  a  thick  layer  of  glass  and 
a  proportionally  thin  layer  of  fluid  or  the  reverse 
is  interposed  between  the  object  and  the  lens  sys- 
tem. 

Anise-oil  was  used  by  Amici  for  the  purpose  of 
increasing  the  exponent  of  refraction ;  by  Spencer 
glycerin  was  employed.  Stephenson  *  desired  an  en- 
largement of  the  aperture  of  the  lens,  not  only  to 
avoid  the  necessity  of  the  correction  of  the  cover- 
glass,  but  also  to  increase  the  power  of  differentia- 
tion. 

The  union  of  these  two  postulates  by  Stephenson, 
their  settlement  by  Abbe,  the  construction  of  lenses 
by  Zeiss,  and  the  introduction  of  this  .system  for 
homogeneous  immersion  by  Koch,f  marked  a  new 
era  for  the  microscopic  side  of  bacteria  investiga- 
tion. 

The  best  fluid  is  the  ethereal  oil  of  cedar,  the  index 
of  whose  refraction  is  the  same  as  crown-glass,  and 
its  index  of  dispersion  differs  only  in  a  slight  degree 
from  crown-glass.  When  somewhat  inspissated,  it  is 
more  convenient  for  use  for  most  optical  purposes. 
By  mixing  other  stronger-refracting  ethereal  oils — 
such  as  oil  of  cloves,  phenol,  and  anise — with  olive-  or 

*  On  a  large  angled  immersion  objective.  "  Journal  of  the  Royal 
Microscopical  Society,"  1878,  p.  61. 

t  "  Untersuchungen  iiber  die  Aetiologie  der  Wtmdinfectionskrank- 
heiten,"  1878. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     37 

castor- oil,  an  immersion-fluid  can  be  made,  which  is 
similar  in  its  refracting  power  to  oil  of  cedar,  or 
which  differs  from  it  to  a  certain  degree. 

In  consequence  of  the  removal  of  the  troublesome 
cover-glass  correction  (attained  by  different  lengths 
of  the  draw-tubes)  which  allows,  in  a  delicate  man- 
ner, a  compensation  for  the  influence  upon  the  aber- 
ration of  the  varying  distance  of  the  picture,  the  ob- 
jectives for  homogeneous  immersion  are  always  ad- 
justed for  a  definite  length  of  the  draw-tube.  On 
this  account  it  is  to  be  noted  that  lengthening  the 
draw-tube  beyond  this  normal  length  acts  in  the  way 
of  a  spherical  over-correction,  shortening  in  the  way 
of  an  under-correction. 

In  using  an  immersion-lens,  one  places  a  drop  of 
oil  on  the  cover-glass,  screws  down  the  draw- tube 
with  the  coarse  adjustment,  or,  if  this  is  lacking, 
brings  it  down  with  a  rotary  motion  by  the  hand,  so 
far  that  the  front  lens  of  the  objective  touches  the  oil 
and  the  picture  begins  to  be  visible.  Then  the  fine 
adjustment  with  micrometer- screw  is  used.  Some 
place  a  drop  of  oil  on  the  front  lens  of  the  objective. 
Others  put  a  drop  not  only  upon  the  objective,  but 
also  upon  the  cover-glass. 

After  use,  the  oil  is  carefully  removed  from  the 
lens  with  a  fine  linen  cloth,  less  satisfactorily  with 
blotting-paper,  and  the  system  is  returned  to  its  case. 
If  the  cover-glass  preparation  is  to  be  preserved, 
then  the  oil  is  soaked  up  with  filter- paper,  and  what 
remains  is  finally  removed  by  chloroform  or  ben- 
zine. 

According  to  histological  tradition,  which  influ- 
enced the  earlier  use  of  the  microscope,  the  magnify- 
ing power  should  be  increased  rather  through  the 
use  of  a  more  powerful  lens  than  a  more  powerful 


38  BACTERIOLOGICAL  INVESTIGATION. 

ocular.  But,  according  to  a  purely  physical  princi- 
ple, the  strength  of  an  ocular,  which  an  objective  will 
allow  with  advantage,  depends  upon  the  angle  of 
aperture  of  the  latter.  The  larger  the  angle  of  aper- 
ture, so  much  the  stronger,  other  things  being  equal, 
can  the  ocular  be.  Our  best  homogeneous  systems 
correspond  to  this  requirement,  namely,  that  one  can 
use  the  same  objective  and  at  the  same  time  employ 
the  most  powerful  oculars. 

In  this  way,  bacteria  without  any  special  prepara- 
tion may  be  observed  between  the  slide  and  cover- 
glass.  One  cause  of  uncertainty  is  here  noticed, 
i.  e.,  almost  all  bacteria  are  in  motion.  This  in  part 
seems  to  be  spontaneous  motion  ;  in  part  a  simple 
Brownian  molecular  movement,  such  as  occurs  in  all 
fine  particles  suspended  in  a  fluid.  In  proportion  to 
the  minuteness  of  the  objects,  these  movements  ren- 
der exact  observation  difficult.  For  this  reason  one 
should  early  eliminate  this,  and  fix  the  forms  of 
micro-organisms  by  narcotizing  them.*  For  this  pur- 
pose, a  particle  of  spirituous  or  dilute  alcoholic  tinct- 
ure of  opium  can  be  added  with  the  point  of  a  needle 
to  a  drop  of  water. 

Moreover,  von  Kecklinghausen  f  has  shown  that 
sma]l,  round,  granular  tissue-detritus,  which  is  so 
easily  confounded  with  bacteria,  can  be  sharply  dif- 
ferentiated by  the  fact  that  bacteria  are  pre-eminent- 
ly homogeneous  granules,  and  are  totally  unaffected 
by  the  action  of  acetic  acid,  glycerine,  or  even  caustic 
soda. 

BaumgartenJ  succeeded  in  making  the  tubercle 

*  Perty,  "Zur  Kenntniss  kleinst»r  Lebensformen,"  1852,  S.  13. 
t  "  Verhandlungen  der  Physikal-Medizin.  Gesellschaft  in  Wurz- 
burg,"  N.  F.,  II.  Bd.,  Heft  4,  1872.    "Sitzungsberichte,"  S.  XII. 
\  "  Centralblatt  f.  d.  med.  Wissenschaft,"  1882,  No.  15. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    39 

bacilli  visible  by  their  resistance  to  a  diluted  solution 
of  caustic  soda,  though  he  could  not  recognize  them 
by  staining  according  to  the  methods  in  use  at  that 
time. 

We  possess  now  more  convenient  means,  both  for 
fixation  and  differentiation.  But  it  would  be  a  seri- 
ous error  not  to  examine  bacteria  unfixed  and  un- 
stained. It  is,  on  the  contrary,  necessary  throughout 
that  the  bacteria  should  be  observed  under  the  most 
natural  conditions  possible  in  order  to  study  their 
motion,  to  follow  the  formation  of  spores  and  their 
germination,  and  to  control  the  forms  according  to 
other  treatment. 

For  this  purpose  we  do  not  use  the  previously 
described  form  of  investigation,  but  employ  the  moist 
chamber.  For  this  the  hollow  slides  A  and  B  (Fig. 
6)  serve.  A  small  drop  (c)  of  the  bacteria-containing 

FIG.  6. 


A,       "c 

fluid  is  placed  upon  a  cover-glass  (b).  The  cover-glass 
is  quickly  reversed,  and,  with  the  drop  now  hanging 
underneath  (e,  B\  is  laid  over  the  hollow  (a)  in  the 
slide,  and  its  edges  are  surrounded  by  vaseline,  wax, 
paraffine,  or  balsam,  in  order  to  prevent  evaporation 
of  the  fluid.  Another  and  a  better  form  is  represent- 
ed in  Fig.  7.  Upon  a  slide,  A  or  B,  a  glass  plate  (b) 


4:0 


BACTERIOLOGICAL  INVESTIGATION. 


is  cemented,  which  has  a  central  circular  opening.    A 
chamber  is  formed  by  laying  a  cover-glass  (a)  over 


FIG.  7. 


A 


e    e 

\   \ 


the  opening.  This  room,  in  place  of  the  almost  half- 
circular  cavity  in  Fig.  6,  is  bounded  by  parallel  walls. 
The  drop  is  prepared  in  a  similar  manner,  and  in  the 
same  way  hangs  within  the  chamber.  These  chambers 
can  be  improvised  if,  instead  of  cementing  a  piece  of 
glass,  a  thin  piece  of  paper  of  corresponding  size, 
with  a  circular  aperture,  is  fastened  upon  an  ordi- 
nary slide.  The  warm  stage  may  be  employed  for  di- 
rect observation  at  higher  temperatures. 


BACTEEIA. 

In  examining  unstained  bacteria  the  diaphragm 
is  used  in  order  to  make  the  structure-picture  clear  ; 
but  small  objects  and  particles,  the  size  of  bacteria, 
imbedded  in  the  tissue  made  visible  in  the  struct- 
ure-picture, are  hidden  by  the  shadows  of  the  struct- 
ure-picture. If  these  particles  are  stained,  and  if 
they  are  of  a  certain  size,  they  will  be  visible  in 
spite  of  the  shadow  ;  but  under  this  size,  notwith- 
standing their  color,  they  are  concealed  by  the 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    41 

shadows.  Therefore  it  is  desirable  to  stain  the 
bacteria  and  to  so  arrange  the  light  that  the  struct- 
ure-picture does  not  further  interfere,  and  that 
the  color-picture,  as  pure  as  possible,  be  presented 
to  the  observer.  Koch  (loc.  cit.)  succeeded  in  pro- 
ducing this  isolation  of  the  color-picture  by  re- 
moval of  the  diaphragm.  In  this  way  so  weak  a 
structure-picture  was  produced  that  the  minutest 
particles  of  the  color-picture  became  distinct.  In 
the  stronger  structure-pictures,  after  the  remov- 
al of  the  diaphragm,  he  used  a  condenser  which 
threw  so  intense  a  cone  of  rays  upon  the  ob- 
ject that  the  diffraction  appearances  were  entirely 
avoided. 

With  such  a  method  of  illumination,  in  which 
the  preparation  is  permeated  in  all  directions  by  the 
penetrating  rays,  only  those  elements  remain  visible 
which  produce  an  absorption  of  the  rays  on  account 
of  their  staining.  Further,  Abbe  in  this  way  has 
shown  that,  although  the  illumination  in  name  re- 
mains central,  yet  the  important  advantages  of  the 
oblique  illumination  are  obtained  through  the  co- 
operation of  the  rays  passing  at  a  greater  inclination 
to  the  axis  of  the  microscope.  On  account  of  this 
co-operation  of  the  oblique  rays  for  the  isolation  of 
the  color-picture,  and  for  the  complete  development 
of  the  capacity  for  differentiation  of  the  oil-immer- 
sion objective  necessary  for  this,  the  condenser  must 
furnish  a  cone  of  light  of  a  size  at  least  equal  to  the 
aperture  of  the  objective,  which  is  made,  according 
to  Stephenson,  with  a  large  angle  of  aperture.  This 
is  as  yet  attained,  in  a  manner  that  fulfills  all  the  re- 
quirements, only  by  an  Abbe  condenser.  In  order 
that  the  structure-picture  can  be  brought  out  in  spite 
of  this  condenser,  an  arrangement  for  interposing  a 


42  BACTERIOLOGICAL  INVESTIGATION. 

diaphragm  is  added,  which  is  furnished  with  open- 
ings of  different  sizes. 

An  Abbe  condenser  belongs  to  the  system  for 
homogeneous  immersion  .in  bacteria  investigation. 
Now  and  then  it  is  useful  to  place  a  drop  of  water  or 
immersion-fluid  between  the  condenser  and  the  under- 
side of  the  slide,  so  that  below  and  above  a  continuous 
union  is  formed.  These  form  immersion-condensers 
which  were  formerly  used  before  the  Abbe  condenser 
was  constructed. 

GENEKAL   PKINCIPLES   OF   STAINING. 

Since  Hartig,  in  1854,  and  Gferlach,  in  1858, 
showed,  by  the  systematic  use  of  carmine  in  histo- 
logical  work,  that  in  employing  coloring-matters  cer- 
tain elements  of  the  tissues  become  more  distinct  and 
can  be  differentiated  from  other  elements,  staining 
has  been  recognized  as  equivalent  to  a  chemical  re- 
action. 

Weigert  *  first  succeeded  in  staining  the  zoogloaa 
masses  of  the  micrococci  with  the  nuclei,  by  the  use 
of  the  nuclei-staining  ammoniac- carmine  solution,  and 
the  subsequent  treatment  with  hydrochloric  -  acid 
glycerine.  This  staining,  it  is  true,  was  first  used 
by  Weigert  as  a  staining  of  cement  substance ;  but 
later  it  was  corrected  by  him,  and  it  was  in  this  way 
shown  that  the  bacteria  can  be  brought  into  view 
by  other  characteristics  than  their  greater  resistance 
to  acids  and  alkalies. 

In  the  following  year  Eberth  and  Wagner  suc- 
ceeded in  staining  micrococci,  but  not  bacilli,  with 
hsematoxylin. 

*  "  Ueber  Bakterien  in  der  Pockenhaut."  "  Centralblatt  f.  d.  med. 
Wissenschaft,"  1871,  No.  49. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    43 

Then  Weigert  *  showed  that  the  micrococci,  espe- 
cially in  zooglcea  masses,  can  be  stained  by  different 
nuclei-staining  materials.  For  this  purpose  he  used 
at  that  time  methyl-violet,  an  aniline-dye  which 
stains  nuclei.  By  the  subsequent  treatment  of  prep- 
arations stained  with  hsematoxylin  (in  which  the 
micrococci  and  nuclei  are  stained  blue),  with  diluted 
caustic  potash  and  strong  acetic  acid,  he  first  suc- 
ceeded in  procuring  an  isolated  staining  of  the  mi- 
crococci. Weigert  f  further  observed  that  the  larger 
bacilli,  which  were  not  stained  by  hsematoxylin, 
could  be  made  visible  by  certain  aniline-dyes.  Soon 
after  this,  Koch:):  found  that  the  bacteria  take  up 
aniline-dyes  with  such  certainty,  and  so  quickly  and 
completely,  that  ' '  these  dyes  can  be  used  as  reagents 
for  differentiation  of  bacteria  from  crystalline  and 
amorphous  precipitates,  or  from  the  smallest  fat- 
drop  or  other  minute  bodies." 

Koch#  then  succeeded  in  obtaining  the  isolated 
staining  of  the  bacteria  by  washing  out  the  section 
with  a  solution  of  carbonate  of  potassium,  by  which 
all  the  elements  except  the  bacteria  were  decolorized. 
Finally  Weigert  ||  obtained  a  double  staining,  when 
he  subsequently  treated  with  picro-carmine  prepara- 
tions that  had  been  stained  with  a  blue  aniline-dye, 
whereby  the  bacteria  appear  blue  and  the  nuclei  red  ; 

*  "  Sitzung  der  Schlesischen  Gesellschaft  fur  vaterlandiscbe  Cul- 
tur,"  vom  10.  December,  1875. 

t  "Berl.  klin.  Wochenscbrift,"  1877,  Nos.  18-19;  und  "Berichte 
fiber  die  Munchener  Naturforscberversammlung,"  1877,  S.  283. 

I  "  Verfahren  zur  Untersuchung,  zum  Conserviren  und  Photo 
graphiren  der  Bakterien."  "Beitrage  zur  Biologic  der  Pflanzen,"  Bd. 
II,  3.  Heft,  1877,  S.  399. 

*  "  Wundinfectionskrankheiten,"  1878,  S.  39. 

||  "Zur  Technik  der  mikroskopischen  Bakterien untersuchungen." 
Vircbow's  "  Arcbiv,"  1881,  Bd.  LXXXIV,  S.  275. 


44  BACTERIOLOGICAL  INVESTIGATION. 

and  Koch*  observed  that  certain  forms  of  bacteria 
stained  differently  from  the  nuclei  and  from  other  bac- 
teria which  were  present  in  the  same  preparations. 

What  coloring-matters  ought  to  be  used  for  stain- 
ing bacteria  ? 

Weigert's  observation  that  the  micrococci,  but  not 
the  bacilli,  are  stained  by  carmine,  and  a  similar  ob- 
servation by  Eberth  and  Wagner  concerning  hsema- 
toxylin,  seemed  to  show,  according  to  Weigert,  that 
there  are  essential  chemical  differences  between  the 
single-group  forms  of  Cohn.  Safranine,  one  of  the 
best  reagents  for  the  staining  of  nuclei,  is  also  of 
more  value  for  micrococci  than  for  other  forms  of 
bacteria.  Further,  Obermeyerf  observed  that  the 
spirilla  are  less  resistant  to  the  action  of  acids  and 
alkalies  than  other  bacteria.  But  these  differences 
are  not  essential,  since  some  micrococci  and  bacilli 
are  less  resistant  to  the  action  of  alkalies  and  acids 
than  others,  and  since  some  bacilli  stain  as  well  with 
hsematoxylin  as  micrococci,  while  others  take  this 
dye  badly. 

However,  the  basic  aniline-dyes  have  shown  them- 
selves to  be  available  staining  materials,  under  all 
conditions,  both  for  the  dried  cover-glass  prepara- 
tions and  for  sections,  so  that  we  assign  to  them  a 
first  rank  among  materials  for  staining  bacteria,  and 
give  to  other  dyes  a  second  place. 

Ehrlich,^  partly  in  -conjunction  with  his  pupils 
Schwarze  and  Westphal,*  undertook  to  classify  the 

*  "Berl.  klin.  Wochenschrift,"  1882,  No.  15. 
t  "Berl.  klin.  Wochenschrift,"  1873,  S.  391. 

t  "Zeitschrift  fur  klin.  Med.,"  Bd.  I,  1880,  S.  653;  und  kleinere 
gelegentliche  Mittheilungen. 

*  Schwarze,   "  Ueber  eosinophile  Zellen,"  Dissert.,  Berl.,  1880. 
Westphal,  "  Ueber  Mastzellen,"  Dissert.,  Berl.,  1880. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    45 

dyes  used  in  microscopic  work.  The  principle  under- 
lying this  theoretical  study  of  dyes  rests  on  the  ob- 
servation that  the  different  elements  of  the  tissues 
and  cells  possess  the  capacity  of  taking  certain  dyes 
only,  or  of  holding  them  with  a  greater  tenacity  than 
other  elements.  This  "  election,"  this  affinity  of  the 
dyes  for  certain  elements,  lends  to  staining  the  value 
of  a  chemical  reaction  ;  or,  more  correctly  (in  the 
want  of  a  precise  chemical  reaction),  shows  to  the 
eye  the  presence  of  differences  otherwise  impercep- 
tible or  distinguishable  only  with  difficulty. 

Many  staining  materials  color  at  first  many  ele- 
ments of  a  tissue  quite  diffusely,  so  that  the  individual 
elements  are  not  recognizable.  If,  then,  certain  de- 
colorizing agents  are  used,  some  of  these  individual 
elements  give  up  their  color,  while,  on  the  other 
hand,  other  elements  retain  it  persistently.  In  this 
way,  by  an  indirect  method,  it  is  possible  to  attain  a 
maximum  staining  of  certain  elements,  while  others 
remain  as  much  as  possible  uncolored.  Ehrlich 
called  this  method,  which  was  first  used  in  another 
way  by  Friedlander,  *  "  the  principle  of  the  maximal 
decolorization." 

Histologically,  one  must  differentiate  in  every  col- 
oring-material two  peculiarities :  first,  the  affinity  to 
certain  elements  ;  and,  secondly,  the  staining  power. 
In  respect  to  the  election  or  affinity  for  certain  ele- 
ments, Ehrlich  divides  the  aniline  colors  into  two 
groups — (a)  the  acid,  (b)  the  basic  aniline-dyes — ac- 
cording as  the  coloring  principle  is  the  acid  or  base- 
color.  In  this  histological  sense  it  is  of  equal  import 
whether  the  acid  in  its  use  acts  as  a  free  acid  or  a 
salt ;  also  whether  the  base  acts  as  such  or  as  a  salt. 

*  "  Studien  fiber  automische  Herzbewegung,"  in  "  Untersuchungen 
aus  dem  physiologiscben  Institut  zu  Wtirzburg,"  I,  1867. 


46  BACTERIOLOGICAL  INVESTIGATION. 

The  acid  aniline  colors  are  divided  into  four 
classes : 

1.  Fluorescin — e.  g.,  fluorescin  and  eosin. 

2.  Nitrogenous  bodies — e.  g.,  martius  yellow,  pic- 
ric acid,  and  aurantia. 

3.  Sulphuric  acid — e.  g.,  tropseolin. 

4.  Primary  dye-acids — e.  g.,  rosol  acid,  alizarin, 
and  purpurin. 

Of  the  basic  aniline-dyes,  the  following  are  found 
to  have  the  most  value :  Fuchsin  (muriate  of  rose- 
anilin),  methyl- violet  (muriate  of  trimethyl  rose-ani- 
lin),  gentian-violet,  methyl-blue,  and  vesuvin.  Of  less 
value  are  methyl-green,  cyanin,  safranin,  magdala, 
and  dahlia.  Of  these,  especially  the  violets  (methyl- 
violet,  gentian- violet,  iodine-violet,  and  dahlia)  have 
sometimes  the  valuable  capacity  of  double  staining  ; 
that  is,  they  stain  certain  elements  in  a  color  varying 
from  the  fundamental  color — e.  g. ,  methyl- violet  does 
not  stain  the  amyloid  substance  violet,  as  it  does  the 
bacteria  and  nuclei,  but  red  ;  methyl-green  stains  the 
nuclei  green,  the  amyloid  substance  violet. 

The  members  of  the  first  group  show  altogether 
the  same  elective  peculiarities — i.  e.,  they  act  as  acid 
dyes  and  stain  all  the  accessible  elements,  but  in  a 
differing  degree.  The  basic  aniline-dyes  also  show 
the  same  elective  peculiarities,  since  they  stain  the 
accessible  elements  in  a  basic  color ;  but  also  in  these 
there  is  a  difference  in  the  intensity  of  the  staining. 
This  intensity  of  the  staining  is  dependent  upon  the 
coloring  power,  and  the  coloring  power  is  conditional 
on  the  fact  that  the  different  coloring-matters  are  re- 
tained in  different  degrees  of  intensity  in  the  tissues 
or  cell-elements,  in  the  presence  of  the  individual 
groups  of  decolorizers,  such  as  alcohol,  acetic  acid, 
and  glycerine.  For  example,  methyl-green  in  a  short 


BACTERIA  AND  MICROSCOPICAL  TECHNIQUE.     47 

time  is  completely  extracted  from  a  preparation  by 
alcohol,  while  vesuvin  is  scarcely  at  all  affected.  In 
respect  to  this  coloring  power,  the  basic  aniline-dyes 
arrange  themselves  in  the  following  order :  Vesuvin, 
bismarck-brown,  and  aniline-brown  theoretically  take 
the  first  place,  because  these  coloring-matters  are  not 
extracted  by  glycerine,  and  they  are  at  the  same  time 
suitable  colors  for  photography.  After  these  follow 
in  a  descending  scale  fuchsin,  methyl-violet,  gentian- 
violet,  and  methyl-blue ;  but  these  should  be  placed 
generally  before  the  brown,  as  they  are  to  most  per- 
sons more  agreeable  and  satisfactory  colors.  The  re- 
maining dyes  have  as  yet  found  no  general  use. 

This  scale,  as  I  will  remark,  to  avoid  misunder- 
standing, has  a  conditional  value,  since  certain  colors 
are  produced  only  when  certain  fluids  are  used  for 
solution  and  when  the  preparations  are  subsequently 
treated  in  a  special  manner,  as  will  be  described  later 
more  in  detail.  At  the  same  time  it  is  not  impos- 
sible that  some  one  of  the  remaining  dyes  may  prove 
to  be  more  valuable  than  these. 

The  basic  aniline  colors  are  soluble  in  water,  and 
for  the  most  part  in  one  or  all  of  the  decolorizing 
agents.  In  use,  a  weak  watery  solution  colors  at  first 
the  intercellular  substance  and  the  cell-body,  while 
the  nuclei  remain  unstained.  Through  the  subse- 
quent treatment  with  alcohol,  glycerine,  or  acetic  acid 
an  inversion  of  the  staining  takes  place,  by  which  the 
elements  previously  colored  become  colorless,  while 
the  previously  colorless  nuclei  are  stained.  In  the 
use  of  the  stronger  solutions  the  staining  follows 
(without  any  discernible  inversion)  directly  and  quick- 
ly ;  and,  in  general,  its  intensity  is  in  proportion  to 
the  concentration  of  the  solution.  In  a  quite  concen- 
trated watery  solution  overs taining  may  occur,  which 


4:8  BACTERIOLOGICAL  INVESTIGATION. 

in  sections  can  be  reduced  to  the  proper  degree  by 
subsequent  decolorization. 

Methyl-blue  alone  does  not  overstain,  according  to 
Ehrlich,*  even  after  a  long  action,  and  it  is  conse- 
quently to  be  used  if  for  a  special  reason  no  decolor- 
izing agents  should  be  employed. 

If  the  dyes  are  dissolved  in  the  decolorizing  agents 
— such  as  absolute  alcohol,  acetic  acid,  or  thick  gly- 
cerine— they  stain  slightly  or  not  at  all.  Instead  of 
using  some  decolorizing  agent  subsequently,  to  re- 
duce the  intensity  of  the  staining  to  the  proper  degree, 
in  preparations  which  have  been  overstained  in  watery 
solutions,  in  many  cases  a  solution  of  the  dye-stuff 
in  a  mixture  of  water  with  alcohol  (Herrmann),  gly- 
cerine (Schaefer),  or  acetic  acid  (Ehrlich)  may  be  used. 

PREPAKATION   OF   STAINING  FLUIDS. 

The  basic  aniline-dyes  are  used  in  the  following 
solutions : 

1.  Concentrated  watery  solutions. 

These  are  either  used  directly  or  after  dilution  to 
the  desired  degree  with  distilled  water.  The  solu- 
tions are  prepared  with  distilled  water  (which  has 
been  previously  boiled),  so  that  an  excess  of  the  col- 
oring-matter remains  undissolved.  They  must  be 
often  filtered.  Only  a  small  quantity  of  these  watery 
solutions  should  be  made  at  a  time. 

2.  Concentrated  alcoholic  solutions. 

The  solution  of  an  excess  of  the  coloring-material 
is  brought  about  in  the  best  way  by  absolute  alcohol, 
or,  in  want  of  this,  by  the  officinal  90  per  cent  spirit  of 
the  Pharmacopoeia. 

In  general,  one  can  calculate  about  20  to  25 
grammes  of  the  dye-stuff  to  100  grammes  of  the  spirit 
*  "  Zeitschrif t  f.  klin  Med.,"  Bd.  II,  1881,  S.  710. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    49 

or  alcohol.  These  solutions  are  kept  prepared,  and 
are  not  used  directly  for  staining,  but  are  mixed  with 
a  certain  amount  of  distilled  water.  In  place  of  con- 
centrated watery  solutions,  these  can  be  used  if  five 
or  six  drops  are  added  to  a  small  watch-glass  of  dis- 
tilled water.  This  mixture  I  shall  briefly  designate 
in  the  future  as  the  diluted  alcoholic  solution. 

3.  Vesuvin,  bismarck-brown,  and  aniline-brown  can 
not  be  used  in  alcoholic  solution,  nor  in  a  watery 
solution,  even  if  filtered  each  time  ;  so  that  a  concen- 
trated solution  in  equal  parts  of  glycerine  and  water 
is  prepared.* 

4.  Alkaline  solutions, 
a.  Weak.    Koch.f 

Concentrated  alcoholic  solution  methyl-blue. .  1    c.  cm. 

Aq.  destil 200    c.  cm. 

10  per  cent  solution  caustic  potash 0*2  c.  cm. 

5.  Strong.J 

Concentrated  alcoholic  solution  methyl-blue .  30  c.  cm. 

Solution  caustic  potash,  1  to  10,000 100  c.  cm. 

5.  Aniline- water  solution  (according  to  Ehrlich).* 
Pure  aniline-oil  in  excess  is  shaken  with  distilled 
water  for  one  half  to  one  minute  (about  5  c.  cm.  of  oil 
with  100  c.  cm.  of  water).  Then,  after  allowing  it  to 
stand  five  minutes,  the  mixture  is  filtered  through  a 
filter  which  has  been  previously  moistened  with  dis- 
tilled water.  The  filtrate  must  be  perfectly  clear,  and 
serves  in  place  of  water  as  a  menstruum.  Since  this 
saturated  aniline  solution  very  quickly  becomes  un- 

*  Koch,  "  Yerfahren  zur  Untersuchung."     "  Beitrage  zur  Biologie 
der  Pflanzen,"  1877,  Bd.  II,  3.  Heft,  S.  5. 

t  "  Berl.  klin.  Wochenschrift,"  1882,  No.  15  ;  "  Mittheilungen  aus 
d.  k.  Gesundlieitsamt,"  1884,  Bd.  II,  S.  5. 
I  "  Mittheilungen,"  1884,  Bd.  II,  S.  439. 

*  "  Deutsche  med.  Wochenschrift,"  1882,  No.  19. 


50  BACTERIOLOGICAL  INVESTIGATION. 

stable,  it  is  better  to  prepare  it  fresh  each  time  that 
it  is  used.  If  it  is  desired  to  make  this  permanent, 
according  to  B.  Fraenkel,  5  to  10  per  cent  of  alcohol 
is  added,  or  3  c.  cm.  of  aniline-oil  is  dissolved  in  7  c.  cm. 
of  alcohol  and  90  c.  cm.  of  distilled  water  is  added. 
Fuchsin,  methyl-violet,  and  gentian-violet  are  the 
best  dyes  for  use  in  this  menstruum. 

In  most  cases,  according  to  Ehrlich,  it  is  more  con- 
venient to  add  a  saturated  alcoholic  solution  of  f  uch- 
sin  or  methyl-violet  to  the  clear  aniline-water  until  a 
distinct  cloudiness  of  the  fluid  is  present,  which  indi- 
cates that  the  fluid  is  saturated  with  the  coloring- 
matter.  For  certain  purposes  the  following  modifi- 
cation of  the  Ehrlich  solution,  according  to  Weigert 
and  Koch,*  recommends  itself  for  common  use,  but 
this  must  be  renewed  after  ten  or  twelve  days,  be- 
cause its  coloring  power  is  generally  diminished : 

Saturated  aniline- water 100  c.  cm. 

Concentrated  alcoholic  solution,   methyl- 
violet,  or  f uchsin 11  c.  cm. 

Absolute  alcohol , 10  c.  cm. 

6.  In  place  of  aniline,  toludin,  prepared  in  the 
same  manner,  can  be  used  as  a  menstruum  (B.  Fraen- 
kel). f  Also  turpentine  (Prior)  ;J  and  a  five  per 
cent  watery  solution  of  carbolic  acid  (Ziehl),*  or 
one  half  per  cent  ammonia  (Weigert)  ||  [Liq.  ammon. 
caust.,  0*5  c.  cm.  ;  aq.  destil.,  90  c.  cm.  ;  alcohol  abs., 
10  c.  cm.  ;  gentian- violet,  2  grm.]. 

For  double  staining,  the  nuclei-staining  carmine 

*  "  Mittheilungen  aus  dera  kaiserlichen  Gesundheitsamt,"  Bd.  II, 
1884,  8.  6. 

t  "  Berl.  k.  Wochenschrift,"  1884,  No.  13. 

I  "  Berl.  k.  Wochenschrift,"  1883,  No.  83. 

*"  Deutsche  med.  Wochenschrift,"  1882,  S.  451;  1883,  S.  12 
tmd  247. 

I  "  Deutsche  med.  Wochenschrift,"  S.  851. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     51 

and  hsematoxylin  *  can  also  be  used ;  the  first  for 
blue  or  violet,  the  latter  for  red-stained  bacteria.  In 
place  of  the  ordinary  nuclei-staining  carmine,  picro- 
carmine  can  be  used  for  preparations  of  bacteria 
stained  blue,  which  stains  the  nuclei  an  intense  red, 
the  fibrillar  substance  of  the  connective  tissue  pink, 
and  the  protoplasmic  substance  a  more  or  less  yellow, 
so  that  a  threefold  staining  results.  Hsematoxylin 
is  best  used  in  the  following  solution  : 

Hsematoxylin 2  parts 

Alcohol 100    " 

Aq.  destil 100    " 

Glycerine 100    " 

Alum,  sulph 2    " 

This  hsematoxylin  stains  micrococci,  many  bacilli, 
and  at  the  same  time  the  zoogtea  masses,  with  the  in- 
tercellular substance.  The  staining  of  the  bacteria  is 
paler  than  that  produced  by  the  blue  or  violet  basic 
aniline-dyes,  which,  on  the  other  hand,  do  not  stain 
the  zooglcea  masses.  In  order  to  obtain  the  threefold 
staining  after  the  bacteria  are  stained  red  by  fuchsin, 
it  is  better  to  stain  the  nuclei  with  hsematoxylin, 
and  then  afterward  stain  the  protoplasm  with  a  satu- 
rated solution  of  picric  acid  or  eosin.  The  rose-red 
eosin  can  be  added  to  the  above  solution  of  hsema- 
toxylin in  the  proportion  of  one  half  per  cent.  (To 
retain  the  yellow  color  of  the  picric  acid,  picric -alco- 
hol and  damar  resin  must  be  used.) 

OTHEE  REAGENTS  AND  APPARATUS. 

Other  solutions  are  often  required : 

(a)  Iodine 1  part 

Potas.  iodid 2  parts 

Aq.  destil 100     " 

*  Of.  die  citirten  Handbtlcher,  besonders  Friedlander. 


52  BACTERIOLOGICAL  INVESTIGATION. 

(b)  In  the  use  of  the  basic  aniline-dyes  it  is  sel- 
dom necessary  to  extract  the  fat.     If  it  is  desirable 
to  do  this,   the  sections  are  first  washed  thorough- 
ly in  absolute  alcohol  (three  to  ten  minutes),  then 
are  transferred   to  a  watch-glass    containing  ether 
and  chloroform  for  a  few  minutes ;  after  this  they 
are  again  placed  in    alcohol,  cleared  up  in  acetic 
acid  (for  the  solution  of  the  coagulated  albumen), 
and  are  then  examined  immediately  or  after  previous 
staining. 

(c)  Nitric  acid,  diluted  in  the  proportion  of  one 
part  of  the  officinal  acid  to  three  or  four  parts  of  wa- 
ter, is  sometimes  used. 

(d)  For  the  removal  of  the  lime  salts,  according  to 
von  Ebner,  the  following  solution  may  be  used,  but 
must  be  often  renewed : 

Ac.  muriat 5  parts 

Alcohol 100      " 

Aq.destil 20      " 

Sodii  chlorid 5      " 

(e)  Acetic  acid  is  employed  in  from  one  half  to  one 
per  cent  solution  for  obtaining  the  maximal  decol- 
orization  and  for  the  examination  of  unstained  bac- 
teria. 

(/)  Chromic  acid  is  used  in  a  one  half  per  cent 
solution,  or  as  Muller's  fluid  : 

Potas.  bichrom 2  parts 

Sodii  sulphat , 1  part 

Aq.  destil 100  parts 

(g)  The  hydrates  of  potassium  or  sodium  in  one 
to  three  per  cent  solutions,  or  a  solution  made  by 
adding  one  to  two  drops  of  the  thirty-three  per  cent 
solution  (so  much  used  in  histology)  to  a  watch-glass 
of  water,  may  be  employed  for  rendering  the  un- 
stained bacteria  visible. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     53 

(7i)  Glycerine  and  alcohol  are  always  to  be  used 
only  in  pure  form  completely  free  from  acid. 

(i)  The  distilled  water  which  is  used  in  bacterio- 
logical work  should  always  be  sterilized  by  boiling 
for  an  hour  over  a  flame  or  in  a  sterilization  appa- 
ratus, since  the  ordinary  distilled  water  always  con- 
tains bacteria  and  their  germs.  All  the  solutions 
used  in  bacteriology  should  be  made  with  sterilized 
distilled  water,  and  all  solutions  and  reagents  must 
be  tested  for  the  possible  presence  of  bacteria.  For 
the  preservation  of  the  solutions,  flasks  with  ground 
stoppers  are  necessary,  and  for  daily  use  small  glass 
bottles  with  hollow-ground  stoppers,  having  above  a 
rubber  cap  and  below  a  capillary  tube,  are  very  con- 
venient. These  capillary  pipettes  serve  to  draw  up 
many  or  few  drops  as  is  desired. 

For  the  preservation  of  the  bacteria  preparations, 
glycerine  can  be  used  only  with  the  brown  dyes,  since 
it  more  or  less  rapidly  extracts  the  other  aniline  col- 
ors. The  glycerine  gelatin  of  Klebs  can  be  used  for 
the  brown  colors. 

The  saturated  solution  of  potas.  acetat.  (1-2)  can  be 
often  advantageously  used  for  the  preservation  of  the 
stained  and  unstained  bacteria ;  but  the  most  univer- 
sally valuable  material  for  mounting  is  Canada  bal- 
sam, which  is  used  most  conveniently  from  a  tube 
(the  artist's  ordinary  paint-tube}  after  having  been 
dissolved. 

For  diluting  Canada  balsam,  turpentine  or  xylol 
must  be  used,  because  chloroform  extracts  the  basic 
aniline-dyes.  For  the  same  reason,  the  precaution 
should  be  taken  not  to  warm  the  balsam.  The  much- 
loved  oil  of  cloves  should  not  be  employed  for  clearing 
up  on  account  of  the  same  objection  ;  but  in  place  of 
it  oil  of  turpentine,  cedar,  or  bergamot  must  be  used. 


5i  BACTERIOLOGICAL  INVESTIGATION. 

In  the  way  of  apparatus  the  following  articles  are 
necessary :  good  slides  and  cover  -  glasses ;  watch- 
glasses  ;  ordinary  porcelain  dishes,  and  those  with 
plain  ground  bottoms ;  crystallization-glasses  of  differ- 
ent sizes,  preferably  two  sizes,  of  which  the  greater  can 
be  used  at  the  same  time  as  a  cover  for  the  smaller ; 
beakers  of  different  sizes ;  small,  square  wire  baskets 
for  holding  test  -  tubes  ;  test  -  tubes  ;  wash-bottles  ; 
graduates  and  pipettes;  a  plate  of  black  glass  or 
porcelain  to  place  under  white  or  unstained  objects  ; 
a  plate  of  white  glass  or  porcelain  to  place  under 
stained  objects;  glass  tubes,  some  drawn  out  to  a 
capillary-point ;  glass  rods,  some  having  from  3  to  5 
cm.  of  platinum  wire  of  different  sizes  melted  into 
one  end  (to  be  used  straight  or  bent  into  a  loop) ; 
spider-forceps  ;  and  many  slide- pincettes.  On  one  of 
these  latter  instruments  the  ends  should  be  bent  out 
somewhat,  and  small  pieces  of  flat  cork  fastened  to 
them  for  taking  hold  of  cover-glasses.  The  slide 
pincettes  can  be  improvised  by  placing  over  ordinary 
pincettes  the  narrowest  possible  ring  of  cork. 

Of  metal  instruments,  it  is  necessary  to  have  some 
teasing-needles,  scissors,  knives,  and  a  wide  spatula 
of  nickel,  steel,  or  platinum,  on  which  the  sections  can 
be  spread  out  in  transferring  them  from  one  fluid  to 
another. 

New  slides  and  cover-glasses  are  first  cleaned  with 
warm  water  and  dried  with  a  linen  cloth.  But  this  is 
not  sufficient,  and  they  must  then  be  carefully  rubbed 
with  a  cloth  which  has  been  dipped  in  spirit.  For 
the  preparation  of  the  flat  hanging-drop  it  is  often 
necessary  to  allow  the  apparently  clean  cover-glass 
to  lie  some  hours  in  absolute  alcohol ;  to  remove  the 
remains  of  the  alcohol  by  ether,  and  then,  finally,  to 
dry  by  evaporation.  Slides  and  cover-glasses  which 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     55 

have  been  used  are  laid  in  concentrated  muriatic,  ni- 
tric, or  sulphuric  acid,  and,  after  cleaning  and  removal 
of  the  acid,  are  treated  with  water  until  all  acid  reac- 
tion has  disappeared,  and  are  then  further  treated  as 
the  new. 

COVER-GLASS  PREPARATIONS. 

After  it  was  observed  that  the  morphological  ele- 
ments in  the  blood,  dried  in  a  thin  layer,  were  not 
materially  altered  by  drying,  Koch  *  first  employed 
these  casual  observations  methodically  in  bacteria 
investigation.  He  spread  out  upon  a  cover-glass,  in 
a  very  thin  layer,  a  drop  of  fluid  containing  bacteria, 
so  that  the  individual  elements  were  brought  very 
nearly  on  the  same  plane.  This  thin  layer  was  then 
fixed  by  simply  drying  in  the  air.  In  order  to  elimi- 
nate the  slight  alteration  produced  by  this,  it  is  nec- 
essary afterward  to  cause  again  a  swelling  up  of  the 
bacteria.  If  the  layer  which  has  been  dried  in  the 
air  remains  too  long  in  the  water  or  glycerine  used  for 
this  purpose,  it  is  entirely  dissolved,  instead  of  only 
partially  swelling. 

If  the  cover-glass  with  the  dried  layer  is  laid  in 
absolute  alcohol,  or  a  one  half  per  cent  solution  of 
chromic  acid,  the  layer  is  rendered  insoluble  in  water 
and  glycerine,  and  no  longer  swells  up.  But  if  the 
layer  which  has  been  made  insoluble  is  put  into  po- 
tassium acetate,  it  swells  sufficiently  without  being 
entirely  dissolved,  and  all  the  forms  seem  to  be  in  a 
natural  condition.  The  solutions  of  the  aniline-dye 
have  the  same  action  and  cause  the  same  swelling 
without  removing  the  layer,  and  at  the  same  time 
stain  the  bacteria. 

*  "  Verfahren  zur  Untersuchung,"  etc.  "  Beitrage  zur  Biologie 
der  Pflanzen,"  1877,  II,  3.  Heft,  S.  399. 


56  BACTERIOLOGICAL  INVESTIGATION. 

In  the  use  of  this  method  in  the  investigation  of 
the  blood,  Ehrlich  *  found  that  the  rapid  drying  pre- 
vented coagulation  of  the  cell-albumen,  and  retained 
the  natural  staining  capacity  of  the  elements.  Only 
the  haemoglobin  was  extracted  by  aqueous  and  gly- 
cerine solutions  of  the  dyes.  But  if  the  prepara- 
tions were  kept  for  a  few  hours  at  a  temperature  of 
115°-125°  C.,  the  elements  of  the  blood,  without  any 
important  alteration  and  without  the  appearance  of 
artificial  products,  retained  their  elective  affinities 
for  dyes.  In  following  up  these  observations,  Koch  f 
discovered  that,  in  place  of  the  fixation  by  alcohol, 
the  application  of  heat  for  only  a  few  minutes  an- 
swered the  same  purpose. 

A  drop  of  the  fluid  containing  bacteria,  either  un- 
diluted or  after  the  addition  of  a  drop  of  distilled 
water  (according  to  the  amount  of  its  morphological 
elements),  is  spread  out  in  a  thin  layer  upon  the 
cover-glass,  by  means  of  a  pointed  scalpel  or  platinum 
wire,  and  the  excess  of  fluid  soaked  up  with  filter- 
paper  ;  or  a  drop  is  placed  upon  one  cover- glass  and 
a  second  is  applied  to  this,  which,  through  its  press- 
ure, spreads  out  the  drop  in  an  even  layer.  If,  then, 
the  two  cover-glasses  are  drawn  apart  with  pincettes, 
we  have  two  similar  preparations.  The  cover-glasses, 
protected  from  dust,  are  allowed  to  remain  until  com- 
pletely dry,  or  they  can  be  dried  in  a  dry-oven  some- 
what more  rapidly.  The  drying  can  also  be  hastened 
by  holding  the  cover-glass  with  the  prepared  side 
upward  high  above  the  gas-flame,  and  moving  it  to 
and  fro  to  prevent  the  direct  action  of  the  flame. 

Upon  the  dried  preparation  a  drop  of  the  staining 

*  "  Zeitschrift  f.  klin.  Med.,"  Bd.  I,  S.  553. 

t  "  Zur  Untersuchung  von  pathogenen.  Organismen."  "  Mitthei- 
lungen  aus  dem  kaiserlichen  Gesundheitsamte,"  1881,  Bd.  I,  S.  1. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     57 

solution  can  then  be  placed  to  stain  the  elements,  but 
only  in  case  the  fluid  is  free  from  albumen  and  the 
staining  follows  quickly,  since,  by  the  prolonged  ac- 
tion of  the  staining  solution,  the  layer  is  completely 
loosened.  If  the  dried  layer  consists  of  an  albumi- 
nous substance,  such  as  blood,  tissue-fluids,  or  sputa, 
on  the  addition  of  the  staining  solution,  precipitation 
occurs. 

On  this  account  it  is  especially  necessary  that  the 
preparation,  after  drying  in  the  air,  should  be  more 
securely  fixed  by  heating.  For  this  purpose  the 
cover-glass  may  be  placed  in  a  drying-box  or  upon 
a  copper  plate.  The  copper  plate  is  laid  upon  a  tri- 
pod, and  one  end  is  heated  by  a  gas-flame,  so  that 
the  different  portions,  at  different  distances  from  the 
flame,  have  varying  degrees  of  temperature.  A  few 
minutes'  exposure  to  a  temperature  of  125°  C.,  or  ten 
to  twenty  minutes  at  110°  C.,  is  sufficient  to  thor- 
oughly dry  bacteria  preparations.  This  may  be  done 
more  conveniently,  and  in  some  cases  also  more  cer- 
tainly, according  to  Koch-Loeffler,  if  the  cover-glasses 
with  the  dried  layer  are  drawn  rather  rapidly  three 
times  through  a  gas-  or  spirit-flame. 

The  reason  for  heating  in  exactly  this  manner,  ac- 
cording to  Koch,*  is  this :  because  in  preparations 
which  have  not  been  heated  the  above-described  pre- 
cipitation occurs,  while  in  preparations  which  have 
been  passed  through  the  flame  only  once  or  twice,  the 
fixation  of  the  elements,  especially  in  those  containing 
much  albumen,  is  not  sufficient  for  all  cases.  In  those 
passed  through  the  flame  three  times,  while  the  forms 
themselves  are  not  materially  altered,  the  capacity 
for  staining  is  retained,  and  the  albuminoid  material 
has  become  so  insoluble  that  precipitation  no  longer 

*  "  Mittheilungen,"  1884,  Bd.  II,  S.  7. 


58  BACTERIOLOGICAL  INVESTIGATION. 

takes  place.  Passing  the  preparations  through  the 
flame  a  yet  greater  number  of  times  destroys  the 
susceptibility  of  the  bacteria  to  the  staining  fluid. 

The  want  of  success  in  making  preparations,  which 
many  beginners  experience,  seems  to  be  especially  due 
to  the  fact  that  the  preparations  are  generally  heated 
before  they  have  been  completely  dried  in  the  air.  If 
the  preparation  still  contains  any  water,  coagulation 
of  the  albuminoid  material  occurs  when  heated,  while 
in  those  completely  free  from  water  this  does  not  hap- 
pen, and  the  albumen  is  rendered  homogeneous  by 
heating. 

The  preparations,  dried  in  the  air  and  then  drawn 
three  times  through  the  flame,  are  now  stained.  The 
cover-glasses,  with  the  prepared  side  upward,  are  laid 
on  a  piece  of  filter-paper,  and,  by  means  of  a  glass 
rod,  a  cap  pipette,  or  the  glass  stopper  with  the  capil- 
lary-tube, a  few  drops  of  a  staining  solution  are 
placed  upon  the  preparation.  The  staining  fluid 
should  remain  about  twenty  minutes,  or  until  it  is 
seen,  by  an  inclination  of  the  cover-glass,  that  the 
preparation  has  already  taken  up  the  color.  If  the 
action  of  the  staining  solution  ought  to  be  prolonged, 
then  it  should  not  be  placed  drop  by  drop  on  the 
cover-glass,  because,  in  drying,  the  staining  solution 
forms  a  ring  of  color  at  the  edge  which  it  is  difficult 
to  remove.  In  this  case  a  sufficient  quantity  of  the 
staining  solution  is  placed  in  a  watch-glass  or  crys- 
tallization-glass, and  the  cover-glass  is  then  taken, 
with  the  prepared  side  downward,  between  the  thumb 
and  index  or  middle  finger,  and  allowed  to  fall  flat 
upon  the  surface  of  the  staining  solution,  so  that  it 
swims  with  the  prepared  side  upon  the  surface  of  the 
fluid.  To  prevent  evaporation,  the  dish  is  covered 
with  a  glass  plate. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     59 

For  the  removal  of  the  excess  of  coloring-matter, 
a  stream  from  a  wash-bottle  is  thrown  obliquely  from 
above  upon  the  cover-glass,  taking  care  not  to  strike 
the  surface  of  the  preparation  directly  ;  or  the  cover- 
glass,  held  in  pincettes,  is  moved  to  and  fro  in  a  beaker 
filled  with  distilled  water ;  or  the  excess  of  the  fluid 
may  be  soaked  up  with  filter-paper,  a  few  drops  of 
water  added  to  be  soaked  up  anew,  and  so  on  until 
none  of  the  coloring-matter  is  given  up  to  the  filter- 
paper.  Then  the  cover-glass  preparation  is  examined 
in  a  drop  of  distilled  water. 

The  upper  side  of  the  cover-glass  is  freed  from 
every  particle  of  water  by  soaking  it  up  with  filter- 
paper,  because  on  it  must  be  placed  a  drop  of  oil  for 
the  homogeneous  immersion-lens. 

If  the  cover-glass  preparations  are  to  be  preserved, 
the  oil  is  removed  with  filter-paper  and  chloroform, 
the  water  by  careful  warming  or  evaporation  (pro- 
tected from  dust),  and  the  dried  preparation  is  di- 
rectly imbedded  in  Canada  balsam. 

Different  dyes  are  used  for  each  variety  of  bacteria, 
since  some  stain  only  the  bacteria ;  others  at  the 
same  time  the  fine  gelatinous  sheath  ;  others  the  cap- 
sule. On  this  account  the  corresponding  pictures 
in  all  the  methods  of  staining  are  not  absolutely 
similar ;  so  that  it  ought  to  be  self-evident  that  al- 
ways, in  comparison,  only  preparations  should  be  used 
which  have  been  treated  in  exactly  the  same  manner. 
These  considerations  must  guide  one  in  the  choice  of 
the  staining  solution.  We  must  therefore  distinguish 
between  staining  for  a  special  purpose — i.  e.,  for  the 
establishment  or  employment  of  coloring  methods 
which  have  been  described  or  proved  to  be  best  in 
particular  cases — and  the  investigation  staining  used 
especially  to  prove  the  presence  of  bacteria. 


60  BACTERIOLOGICAL  INVESTIGATION. 

Since  in  cover-glass  preparations  almost  all  bac- 
teria can  be  stained  by  watery  solutions  of  the  basic 
aniline-dyes,  saturated  watery  solutions  or  the  equal- 
ly valuable  alcoholic  solutions  are  first  employed. 
The  saturated  watery  solutions  have  for  this  testing 
an  advantage,  because  all  basic  aniline-dyes  are  known 
to  be  applicable ;  so,  with  a  few  preparations,  the  dif- 
ferent colors  can  be  tried. 

If  no  bacteria  come  to  view  in  this  way,  notwith- 
standing their  supposed  presence,  then  aniline-water, 
with  methyl- violet  or  fuchsin,  is  used,  or  the  stronger 
alkaline  solution  of  methyl-blue.  The  trial  examina- 
tion as  to  the  presence  of  bacteria  resolves  itself,  in 
the  larger  number  of  cases,  into  the  following  pro- 
cedure : 

1.  Drying  in  a  thin  layer. 

2.  Fixation  by  passing  the  cover-glass  three  times 
through  the  flame. 

3.  Staining  by  placing  a  few  drops  of  a  watery  or 
dilute  alcoholic  solution  of  a  basic  aniline-dye  upon 
the  preparation. 

4.  Removal  of  the  excess  of  the  coloring-matter 
by  washing  or  soaking  up  with  filter-paper. 

5.  Examination  in  a  drop  of  distilled  water. 

For  the  isolated  staining  of  bacteria  in  cover-glass 
preparations  they  can  be  laid  for  about  one  minute 
in  a  half -saturated  solution  of  potassium  carbon- 
ate ;  or,  if  they  are  stained  in  aniline-water-gentian- 
violet,  the  remaining  elements  can  be  decolorized  ac- 
cording to  the  method  of  Gram.*  The  stained  cover- 
glass  preparations  are  for  this  purpose  laid  for  about 
one  minute  in  the  solution  of  potassium  iodide  (vide 
page  51),  and  then  placed  in  absolute  alcohol  until 

*  "  Ueber  die  isolirte  Farbung  der  Schizomyceten."  "  Fortschritte 
der  Medizin,"  II,  1884,  No.  6. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     61 

they  appear  decolorized.     The  alcohol  is  soaked  out 
and  the  preparation  examined  in  water. 

For  double  staining  the  cover-glass  preparations, 
after  being  decolorized  according  to  the  Gram  meth- 
od, they  can  be  taken  from  the  alcohol  and  placed  in 
a  weak  watery  solution  of  vesuvin.  Then  the  bac- 
teria remain  blue,  often  almost  blue-black,  while  the 
nuclei  are  stained  brown.  The  preparations  stained 
red  or  blue  can  also  afterward  be  stained  with  car- 
mine or  hsematoxylin ;  yet  this  double  staining  has 
much  less  value  in  the  cover-glass  preparations  than 
in  sections. 

EXAMINATION   FOR  TUBERCLE   BACILLI   IN   SPUTUM. 

These  preparations  can  be  stained  according  to 
the  Gram  method;  but  by  this  both  the  tubercle  bacilli 
and  other  bacteria  are  stained  blue  in  contrast  with 
the  brown  nuclei.  For  the  differential  diagnosis  this 
is  not  sufficient,  and  for  this  purpose  the  principle 
established  by  Koch  must  be  exclusively  observed 
— i.  e.,  that  the  tubercle  bacilli  should  be  stained  in 
a  different  color  from  other  bacteria  and  the  nuclei. 
Koch  succeeded  in  doing  this,  in  preparations  stained 
for  twenty-four  hours  in  a  weak  alkaline  solution  of 
methyl-blue,  and  then  placed  for  a  short  time  in  a 
watery  solution  of  vesuvin.  In  this  way  the  tubercle 
bacilli  (and  the  bacilli  of  leprosy)  are  stained  blue ; 
all  other  bacteria  and  nuclei,  brown.  After  this  im- 
portant principle  was  discovered,  Ehrlich  showed 
that  aniline-water  was  still  a  better  agent  for  increas- 
ing the  intensity  of  the  color,  and  that  in  the  prepara- 
tions stained  with  aniline-water  colors  the  tubercle 
bacilli  withstood  decolorization  by  nitric  acid,  while 
all  other  bacteria  were  decolorized  by  this  mineral 
acid.  But  the  preparations  can  not  be  left  so  long  in 


62  BACTERIOLOGICAL  INVESTIGATION. 

the  acid  that  complete  decolonization  occurs,  because 
then  also  many  or  all  of  the  tubercle  bacilli  are  de- 
colorized. They  should  remain  in  the  acid  until  the 
red  (fuchsin)  or  blue  (methyl- violet)  hue  has  changed 
into  a  yellow-red  or  greenish  blue.  At  this  stage  the 
preparations  are  placed  in  water,  and  again  a  red  or 
blue  color  appears.  By  the  action  of  the  acid  the 
simple  acid  union  (red  or  blue)  is  changed  into  a 
triple  acid  (yellow-red  or  blue-green),  and,  by  the 
addition  of  the  water,  the  triple  acid  union  is  de- 
stroyed and  the  red  or  blue  hue  reappears.  The 
preparations  decolorized  by  the  acid  are  not  washed 
in  water,  but  in  50  or  60  per  cent  alcohol ;  then  they 
are  stained  in  a  dilute  solution  of  methyl-blue  (or 
vesuvin).  After  washing  away  the  methyl-blue  (or 
vesuvin)  the  preparations  are  examined  in  water,  or, 
after  removal  of  the  water,  preserved  in  Canada 
balsam. 

After  this  whole  procedure,  the  tubercle  bacilli 
retain  their  red  or  blue  color,  and  are  easily  recog- 
nized among  the  other  elements.  Aside  from  this 
differential  diagnostic  action  of  the  double  staining, 
the  subsequent  staining  in  another  color  has  an  ad- 
vantage by  affording  an  easier  examination  of  the 
specimens. 

Concerning  the  choice  of  the  material  containing 
bacteria,  it  is  to  be  noted  that  the  cheesy  masses  are 
to  be  spread  out  thin  with  a  sterilized  scalpel.  Nod- 
ules of  tubercle  must  be  crushed  with  a  scalpel  or 
between  two  scalpels,  and  then  be  pressed  flat  upon 
the  cover-glass.  The  tough,  yellowish  masses  from 
the  sputum  are  used.  One  of  these  particles  is  taken 
and  spread  out  in  a  thin  ]ayer  on  the  cover-glass,  or 
flattened  by  pressing  one  cover-glass  upon  another, 
so  that,  after  separating  the  two  cover-glasses  with 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     63 

pincettes,  two  preparations  are  obtained.  The  entire 
method  is,  according  to  Koch  (after  the  adoption  of 
the  previously  described  aniline-water  staining  of 
Ehrlich),  briefly  as  follows : 

1.  Pass  the  dried  cover-glass  preparations  three 
times  through  the  flame. 

2.  Stain  with  the  Weigert-Koch  solution  of  methyl- 
violet  or  fuchsin  for  twelve  hours. 

3.  Treat  with  dilute  nitric  acid  (1  to  3  or  4)  for  a 
few  seconds. 

4.  Wash  in  a  60  per  cent  solution  of  alcohol  by  a 
to-and-fro  motion. 

5.  Stain  in  a  dilute  solution  of  vesuvin  or  methyl- 
blue. 

6.  Wash  and  examine  in  water  or  mount  in  bal- 
sam. 

This  method  is  the  best  thus  far  discovered,  and 
serves  as  a  control  in  all  doubtful  cases. 

For  the  differential  diagnostic  decolorization  and 
subsequent  staining  of  preparations  of  tubercle  ba- 
cilli, the  following  reagents  have  been  used : 

1.  Other  aniline-dyes  (vesuvin  by  Koch). 

2.  Acids  (nitric  by  Ehrlich,  hydrochloric  by  Orth, 
acetic  by  Petri). 

3.  Acid  alcohol  (weak  nitric  by  Eindfleisch,  and 
hydrochloric  by  Orth). 

B.  Fraenkel  combined  these  three  variations  by 
preparing  an  acid-alcoholic  solution  of  methyl-blue 
and  of  vesuvin. 
a.  For  blue : 

Alcohol 50  parts. 

Water 30    u 

Nitric  acid 20    u 

Methyl-blue  to  saturation. 
To  be  filtered. 

5 


64:  BACTERIOLOGICAL  INVESTIGATION. 

b.  For  brown : 

Alcohol 70  parts. 

Nitric  acid 30     " 

Yesuvin  to  saturation. 

To  be  filtered. 

For  the  use  of  these  solutions  the  following 
method  of  Fraenkel  is  recommended :  About  5  c.  cm. 
of  aniline-water  are  heated  in  a  test-tube  to  boiling 
and  poured  out  in  a  watch-glass ;  to  this  hot  aniline- 
water  a  concentrated  alcoholic  solution  of  fuchsin  or 
methyl-violet  is  added,  drop  by  drop,  until  the  solu- 
tion of  the  dye  assumes  a  cloudy  appearance.  Upon 
this  warm  solution  the  cover-glass  preparations  are 
allowed  to  swim,  and  even  in  two  or  three  minutes 
most  of  the  tubercle  bacilli  are  stained ;  but,  as  a 
precaution,  they  should  be  left  five  or  ten  minutes. 
From  this  staining  solution,  the  preparations  stained 
red  or  blue  are  passed  into  the  blue  or  brown  acid- 
alcohol  solution.  After  remaining  one  or  two  min- 
utes in  the  latter,  the  preparations  seem  colored  and 
are  washed  in  water  or  50  per  cent  alcohol,  to  which 
one  half  per  cent  acetic  acid  has  been  added,  and  are 
then  examined  in  water. 

Those  who  prefer  the  hydrochloric  acid  of  Orth 
can  employ  the  following  method  of  Kaatzer :  stain 
as  before,  and  then  decolorize  with  a  mixture  of— 

Alcohol  (90  per  cent) 100  c.  cm. 

Water 20  c.  cm. 

Concentrated  hydrochlor.  acid    20  gtt. 
wash  with  90  per  cent  alcohol  to  remove  the  acid,  and 
then  stain  with  a  concentrated  watery  solution  of 
methyl-blue  or  vesuvin. 

In  sputum,  according  to  Celli  and  Guarnieri,  some- 
times very  fine  fat-crystals  are  found,  which  react  to 
the  staining  almost  as  the  tubercle  bacilli,  "pseudo- 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.     65 

bacilli, "  but  which,  on  careful  examination,  are  not  to 
be  confounded  with  them,  on  account  of  their  vary- 
ing size  and  because  they  are  dissolved  by  ether  and 
chloroform. 

The  published  modifications  of  the  Ehrlich  meth- 
od, founded  on  the  Koch  principle,  are  so  numerous, 
but  without  anything  having  been  added,  that  I  must 
on  this  account  simply  refer  to  some  of  the  compre- 
hensive descriptions,  *  and  content  myself  with  giving 
the  underlying  method  of  Koch  (which  is  now  best 
employed  according  to  Ehrlich- Weigert-Koch),  and 
two  practical  modifications  of  it. 

In  these  methods  the  bacilli  of  leprosy  react  as 
the  tubercle  bacilli,  from  which,  moreover,  they  are 
morphologically  not  easy  to  differentiate.  The  differ- 
ential diagnosis  by  staining  is  founded  on  this  fact : 
that  the  bacilli  of  leprosy  do  not  stain  so  deeply  as 
the  tubercle  bacilli,  and  that,  in  respect  to  the  ease 
with  which  they  give  up  the  dye,  they  stand  between 
these  and  most  other  forms  of  bacteria.  According  to 
Baumgarten,f  the  dried  cover-glass  preparations  are 
allowed  to  float  for  six  or  seven  minutes  in  a  dilute 
alcoholic  solution  of  f  uchsin  (5  or  6  drops  of  a  con- 
centrated alcoholic  solution  in  a  watch-glass  of  dis- 
tilled water),  decolorized  fifteen  seconds  in  acid  alco- 
hol (1  part  nitric  acid  to  10  parts  alcohol),  washed  in 
distilled  water,  afterward  stained  in  a  watery  solu- 
tion of  methyl-blue,  washed  again,  and  examined  in 
water. 

*  Kaatzer,  "  Die  Technik  der  Sputumuntersuchung  auf  Tuberkel- 
Bacillen,"  1884.  B.  Fraenkel,  "Ueber  die  Farbung  des  Koch'schen 
Bacillus,"  "  Berl.  klin.  Wocbenschrift,"  1884,  No.  13.  Baumgarten, 
"Beitrage  zur  Darstellungsmethode  der  Tuberkel-Bacillen,"  "Zeit- 
schrift  fur  wissenschaftliche  Mikroskopie,"  I,  1884,  S.  51. 

t  "  Ueber  UntersuchuDgsmethoden  zur  Unterscheidung  von  Lepra- 
und  Tuberkel-Bacillen,"  ibid.,  S.  367. 


66  BACTERIOLOGICAL  INVESTIGATION. 

The  bacilli  of  leprosy  seem  then  as  red  rods  upon 
a  blue  ground,  while  the  tubercle  baciDi  during  this 
time,  by  this  treatment,  have  as  yet  taken  up  no  color. 

The  reaction  of  the  tubercle  bacilli,  both  in  the 
method  of  staining  discovered  by  Koch  with  its  differ- 
ent modifications,  and  in  the  method  of  Baumgarten 
(the  recognition  of  these  bacteria  in  unstained  condi- 
tions), seems  at  first  to  separate  these  bacilli  qualita- 
tively from  all  other  bacteria.  Further  studies,  how- 
ever, have  shown  that  these  differences  are  not  quali- 
tative, but  are  especially  quantitative.  The  tubercle 
bacilli  are  stained  with  the  greatest  difficulty ;  but 
they  retain  the  color  persistently.  It  has  been  shown 
since  by  Lichtheim,*  and  especially  by  the  work  of 
Baumgarten,  previously  cited,  that  the  tubercle  bacilli 
in  dried  cover-glass  preparations  are  stained  in  about 
one  hour,  both  in  dilute  alcoholic  and  strong  aqueous 
solutions  of  methyl-violet,  gentian- violet,  and  f  uchsin, 
or  by  simultaneous  warming,  in  about  five  minutes. 
(For  sections  the  time  required  is  about  twelve  hours, 
or  about  ten  minutes  with  elevation  of  temperature.) 

These  stainings  withstand  the  decolorization  by 
acids  for  some  time,  although  after  a  longer  action  of 
the  same  the  tubercle  bacilli  are  also  decolorized. 
The  tubercle  bacilli  remain  unchanged  after  treatment 
with  carbonate  of  potassium,  as  do  other  bacteria.  If 
the  stained  cover-glass  preparations  are  laid  for  de- 
colorization for  about  one  minute  (sections,  five  min- 
utes) in  alcohol,  and  afterward  for  five  minutes  (sec- 
tions, fifteen  to  twenty  minutes)  in  a  concentrated 
aqueous  solution  of  vesuvin  or  methyl-blue,  a  double 
staining  is  obtained.  Therefore,  neither  the  addition 
of  the  alkali  nor  the  aniline-water  is  absolutely  essen- 

*  "  Zur  diagnostischen  Verwerthung  der  Tuberkel-Bacillen." 
"Fortschritte  der  Medizin,"  1883,  S.  1. 


BACTERIA   AND  MICROSCOPICAL   TECHNIQUE.     67 

tial  to  the  staining ;  the  reaction  to  acids  is  not  a 
qualitative  differentiation  from  other  bacteria,  and 
the  use  of  the  acid  is  not  entirely  necessary  for  ob- 
taining the  double  staining. 

A  satisfactory  explanation  of  the  theory  of  stain- 
ing, which  seems  to  lie  so  near,  has  not  yet  been  dis- 
covered, on  account  of  the  varying  quantitative  re- 
action of  the  tubercle  bacilli.  The  possibility  of  a 
better  understanding  is  offered  by  the  following  fact : 
The  addition  of  an  alkali,  as  well  as  the  feebly  alka- 
line aniline-oil,  renders  the  staining  easier.  Other 
aromatic  bodies  and  ammonia  act  in  a  similar  man- 
ner. The  addition  of  an  acid  to  the  aniline-water 
does  not  arrest  its  action,  so  that  probably  the  favor- 
able action  of  carbolic  acid  is  reduced  to  the  aromatic 
element,  and  is  effectual  in  a  similar  manner  in  spite 
of  the  acid  reaction. 

In  addition,  Gribbs  *  showed  that  in  the  simulta- 
neous action  of  two  aniline-dyes  the  bacilli  are  stained 
differently  from  the  other  elements,  while  by  another 
method  these  are  stained  in  both  of  the  dyes.  A  so- 
lution of  3  c.  cm.  of  aniline-oil  in  15  c.  cm.  of  spir- 
its is  added  slowly  to  2  grammes  of  fuchsin  and  1 
gramme  of  methyl-blue,  and,  after  the  solution  of 
the  dyes,  15  c.  cm.  of  water  are  added.  This  solution 
is  warmed,  the  cover-glass  preparation  is  laid  upon 
it  for  five  minutes,  and  then  washed  in  spirits  till 
color  is  no  longer  given  up.  The  bacilli  then  appear 
red  upon  a  blue  ground.  This  method  is,  unfortu- 
nately, not  sufficiently  reliable. 

EXAMINATION   OF  BLOOD   FOR  BACTERIA. 

The  examination  of  blood  for  bacteria  offers  very 
great  difficulty,  because  in  the  normal  blood  within 

*" Lancet,"  1883,  p.m. 


68  BACTERIOLOGICAL  INVESTIGATION". 

the  vessels,  and  in  the  normal  disintegration  of  the 
healthy  blood,  granular  elements  are  present,  or  are 
formed,  which,  under  certain  pathological  conditions, 
in  anaemic  states  and  in  fever,  are  increased  in  num- 
ber, and  can  be  easily  mistaken  for  micrococci.  They 
have  already  been  often  confounded  with  micrococci, 
and  are  almost  daily  mistaken  for  them — e.  g.,  the  re- 
nowned syphilitic  corpuscle,  and  the  so-called  organ- 
isms of  the  venom  of  serpents.  Here  belongs  also  much 
of  what  has  been  spoken  of  as  the  development  of  bac- 
teria from  nitrogen  molecules,  from  microzymen,  or 
from  the  anamorphosis  of  protoplasm.  An  exact  study 
of  these  granules  of  the  blood  is  on  this  account  an 
indispensable  desideratum  in  bacteria  investigation. 

These  granules  form,  further,  a  constituent  part  of 
the  cellular  elements  of  the  blood,  and  on  this  ac- 
count again  are  of  interest  in  aetiology,  because  there 
are  parasites  which  are  similar  to  the  amoeboid  cells 
— e.  g.,  those  monads  found  by  Lewis  in  the  blood  of 
rats,  by  Koch  in  the  blood  of  marmots.  The  ele- 
ments of  the  blood,  which  directly  or  through  their 
granules  may  be  confounded  with  micro-organisms 
(with  the  exception  of  the  red  blood-corpuscles,  and 
the  products  of  their  disintegration),  are  divided,  ac- 
cording to  Ehrlich,*  into — 
.  Lymphoid  elements. 

(a)  Small  lymph-cells.     (5)  Large  lymph-cells. 

2.  Myeloid  cells  (eosinophile). 

3.  Undetermined  (spleen  and  [or]  marrow). 

(a)  Large   mononuclear    cells.      (5)   Transitional 
forms,     (c)  Polynuclear. 

*  Cfr.  the  work  of  Ehrlich,  Westphal,  Schwarze,  and  Spilling, 
"Ueber  Blutuntersuchungen  bei  Leukamie,"  Dissert.,  Berlin,  1880; 
and  Einhorn,  "  Ueber  das  Verhalten  der  Lymphocyten  zu  den  weis- 
sen  Blutkorperchen,"  Dissert.,  Berlin,  1884. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.  69 

The  small  lymphoid  elements  are  somewhat  small- 
er than  the  red  blood-corpuscles,  possess  a  very  large 
nucleus,  so  that  there  is  very  little  or  no  protoplasm 
to  be  seen.  The  large  lymph-elements  are  a  further 
development  of  the  first,  and  are  only  to  be  differen- 
tiated from  them  in  this,  that  they  possess  around 
the  large  nuclei  a  distinct  border  of  protoplasm.  The 
myeloid  elements  are  large,  round  cells,  with  large, 
oblong  nuclei.  The  mononuclear  cells  are  about 
three  times  the  size  of  the  red  blood- corpuscles,  and 
possess  round  or  oval  nuclei  of  large  size,  and  a  con- 
siderable mass  of  protoplasm.  The  mononuclear 
transitional  forms  are  to  be  differentiated  from  these 
cells  only  in  this,  that  the  nuclei  are  no  longer  round 
or  oval,  but  have  become  indented.  The  polynuclear 
elements  are  somewhat  smaller,  but  still  are  always 
larger  than  the  red  blood- corpuscles,  and  their  nuclei 
show,  as  a  further  differentiation,  a  polymorphous 
form.  These  are  the  true  white  blood-corpuscles. 
The  granular  elements,  or  granules,  which  are  pres- 
ent in  the  cells,  and  which  become  free  in  the  de- 
struction of  the  same,  are  divided  with  respect  to 
their  reaction  to  aniline -dyes. 

/  The  a,  or  eosinophile  granule,  is  coarsely  spher- 
ical, strongly  refracting,  and  can  be  stained  in  all  the 
acid  aniline-dyes.  It  is  present  in  the  myeloid  ele- 
ments, seldom  in  the  normal  blood,  and  its  number 
is  greatly  increased  in  leucsemic  processes. 

The  /3,  or  amphophile  granule,  is  found  especially 
in  the  marrow,  very  often  in  the  leucocytes  in  the 
blood  of  rabbits  and  guinea-pigs,  and  can  be  stained 
by  acid  and  basic  aniline-dyes. 

The  7,  or  basophile  plasma  cell  granule,  can  be 
stained,  like  the  bacteria,  by  the  basic  aniline-dyes. 
These  granules  are  coarse,  slightly  refracting,  almost 


70  BACTERIOLOGICAL  INVESTIGATION. 

completely  wanting  in  normal  human  blood,  increased 
in  leucaemic  processes,  and  are  present  normally  in  the 
blood  of  the  lower  animals,  especially  the  white  rat. 

The  S,  or  basophile  granule,  is  fine,  and  can  be 
stained  in  basic  aniline-dyes,  and  forms  a  constituent 
part  of  the  large  mononuclear  elements. 

The  e,  or  neutrophile  granule,  is  very  fine,  and 
fills  the  polynuclear  elements  of  the  human  blood 
quite  thickly,  is  present  sparsely  in  the  transitional 
forms,  and  very  seldom  in  the  mononuclear  ele- 
ments. It  can  be  stained  by  the  neutral  dyes. 

Without  recourse  to  staining,  these  granules,  as 
a  whole,  and  also  the  products  of  the  disintegration 
of  the  red  blood-corpuscles,  may  be  confounded  with 
micrococci.  By  systematic  staining  with  the  aniline- 
dyes,  the  a,  y3,  and  e  granules  can  be  excluded.  An 
error  is  then  possible  only  with  the  y  and  8  granules, 
because  these  are  stained  in  the  basic  aniline- dyes, 
the  same  as  the  bacteria.  These  last,  on  account  of 
their  fine  grain,  can,  with  comparative  ease,  be  differ- 
entiated from  micrococci,  and  have  not,  as  yet,  been 
confounded  with  them.  The  plasma  cell  granules,  on 
account  of  their  medium  size,  come  so  near  to  the 
known  forms  of  cocci,  that  not  only  the  individual 
free  granules  in  the  blood  have  been  considered  as 
cocci,  but  even  the  same  so-called  plasma  cells  in  the 
tissues  have  been  described  as  colonies  of  cocci. 
They  can,  on  purely  morphological  grounds,  be  dif- 
ferentiated from  them  in  this  way,  viz.,  that  they  do 
not  all  have  the  symmetrical  appearance  of  the  cocci, 
but  present  the  greatest  differences  in  the  size  of  the 
granules. 

If  it  is  desired  to  examine  the  blood  for  bacteria, 
a  small  drop  is  rapidly  spread  out  in  a  thin  layer, 
dried,  fixed,  and  then  passed  three  times  through  the 


BACTERIA  AND  MICROSCOPICAL  TECHNIQUE.    71 

flame,  and  then  stained  in  the  ordinary  manner.  In 
such  preparations  the  bacteria  are  sufficiently  stained, 
but  not  the  granules. 

The  small  drop  of  blood,  at  most  the  size  of  a  pin- 
head,  must  be  taken  with  the  greatest  care.     In  case 
of  a  haemorrhage,  a  small  drop  is  taken  with  a  steril- 
ized platinum  wire,  or,  for  obtaining  the  blood,  the 
skin  may  be  pricked  with  a  previously  sterilized 
needle,  the  end  of  the  finger  being  the  best  spot. 
The  skin  is  first  cleansed  with  a  brush  and  soap,  and 
then  washed  with  a  solution  of  corrosive  sublimate, 
1  to  1,000.     The  sublimate  is  removed  with  alcohol, 
the  alcohol  with  ether,  and  the  last  allowed  to  evapo- 
rate.   The  first  drop  of  blood  which  wells  up  is  re- 
moved with  a  sterilized  platinum  wire,  and  the  fol- 
lowing drop  used,  a  cover-glass  being  lightly  pressed 
upon  it  with  the  pincettes,  without  coming  in  contact 
with  the  surrounding  skin.     Upon  this  first  cover- 
glass  a  second  is  laid,  which,  by  pressure,  spreads 
out  the  blood  in  a  thin  layer,  in  which  the  elements 
are  not  materially  altered.     The  two  cover-glasses  are 
then  drawn  apart  by  pincettes,  so  that  two  cover-glass 
preparations  are  obtained.     Some  of  the  cover-glasses 
are  used  as  above  in  the  examination  for  bacteria, 
after  the  layer  has  been  dried  in  the  air,  and  heated 
only  for  a  short  time  ;  but  others  are  heated  for  one 
hour  at  a  temperature  of  120°  C.,  and  then  treated 
with  a  basic  aniline-dye,  in  order  to  study  more  ex- 
actly the  basophile  granules. 

Other  preparations  for  the  determination  of  the 
eosinophile  elements  are  treated  with  the  acid  aniline- 
dyes  after  heating  a  short  time,  and  also  after  an 
hour's  heating.  A  mixture  is  made  of  the  yellow, 
red,  and  black  dye-stuffs  of  the  strongest  staining 
power,  each  of  which  alone  stains  all  of  the  acid- 


72  BACTERIOLOGICAL  INVESTIGATION. 

forming  elements,  and  both  the  simultaneous  action 
and  the  elective  staining  are  valuable,  since  the  three 
eosinophile  elements  are  stained  at  the  same  time  in 
different  colors.  One  part  of  a  saturated  glycerine 
solution  of  aurantia  is  diluted  with  two  parts  of 
glycerine;  then  aniline-black  (sulphate  of  indolin) 
and  eosin  are  added  in  excess,  and,  by  long  shak- 
ing, dissolved  to  saturation.  This  saturated  glyc- 
erine solution  stains  all  the  parts  containing  hae- 
moglobin intense  orange,  the  nuclei  gray-black  to 
black,  and  the  eosinophile  granules  red  to  red- 
black. 

For  staining  the  neutrophile  granules  the  neutral 
dyes  are  used,  which  are  formed  by  the  union  of  the 
basic  and  acid  dyes — e.  g.,  if  acid-fuchsin  and  orange 
(G)  are  mixed  with  the  basic  methyl-green.  Accord- 
ing to  Ehrlich,  125  c.  cm.  of  a  saturated  aqueous  solu- 
tion of  orange  are  mixed  with  125  c.  cm.  of  a  saturated 
solution  of  acid-fuchsin  in  20  per  cent  alcohol ;  75  c. 
cm.  of  absolute  alcohol  are  then  added,  and  after- 
ward 125  c.  cm.  of  a  saturated  aqueous  solution  of 
methyl-green  with  shaking. 

The  solution  remains  standing  for  some  time,  and 
both  a  precipitation  occurs  and  a  film  forms  on  the 
surface.  In  order  to  obtain  the  solution  quite  clear, 
a  pipette  is  introduced  into  the  middle  of  the  solution 
and  a  quantity  of  the  clear  fluid  is  withdrawn.  The 
pipette  as  well  as  the  vessel  must  be  absolutely  dry,  or 
otherwise  a  further  cloudiness  appears.  By  this  mixt- 
ure the  haemoglobin  is  stained  yellow  to  orange,  the 
nuclei  green,  neutrophile  granules  violet,  and  the 
eosinophile  dark  gray  with  a  tinge  of  blue. 

For  the  demonstration  of  the  cells  in  the  blood 
the  following  mixture  is  used :  * 

*  Ehrlich,  "Deutsche  med.  Wochenschrift,"  1883,  No.  46." 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.  73 

Water. . .. 100  c.  cm. 

Glycerine 100    " 

Absolute  alcohol 100    " 

Hsematoxylin 1-2  gnn. 

Eosin 1     « 

Glacial  acetic  acid 10  c.  cm. 

Alum  to  saturation. 

The  red  blood-corpuscles  show  an  intense  red  color, 
the  nuclei  of  the  lymphoid  and  polynuclear  cells  are 
stained  intensely  blue,  and  the  nuclei  of  the  mono- 
nuclear  cells  bluish  green.  The  protoplasm  of  the 
large  lymphoid  and  polynuclear  cells  is  reddish,  that 
of  the  mononuclear  cells  dark  green. 

The  importance  of  this  method  of  examination  for 
micro-organisms  in  the  blood  has  been  lately  pointed 
out  by  Koch ;  *  but  it  has  not  as  yet  received  neces- 
sary attention.  The  descriptions  given  here  are  all 
the  more  needful  because  our  good  text-books  on 
histological  technique  do  not  sufficiently  treat  this 
subject,  for  the  reason  that  the  original  works  are  so 
difficult  of  access. 

METHODS  OF  STAINING  THE  FLAGELLA. 

The  flagella  (Fig.  4 ;  7,  9,  14,  16),  which  become 
visible  in  the  hanging- drop  at  one  or  both  extremi- 
ties of  the  bacteria  by  forming  an  eddy,  can  be  best 
stained  in  dried  cover-glass  preparations,  according 
to  Koch,f  by  the  addition  of  a  concentrated  aqueous 
solution  of  campechianum.  The  flagella  are  stained 
brown,  but  the  staining  in  this  manner  is  not  durable. 
On  this  account  the  stained  preparations  are  laid  for 
some  time  in  a  5  per  cent  solution  of  chromic  acid  or 

*  "  Mittheilungen,"  Bd.  I,  1881,  S.  7. 

t  "  Verfahren  zur  Untersuchung,  etc."  "Beitrage  zur  Biologie  der 
Pflanzen,"  1877,  Bd.  II,  3.  Heft,  S.  419. 


74  BACTERIOLOGICAL  INVESTIGATION. 

in  Mailer's  fluid.  Then  there  is  formed  an  insoluble 
brownish  black  union  of  the  extract  of  hsematoxylin 
with  the  chromic  acid.  After  washing,  these  prepa- 
rations can  be  directly  preserved  in  glycerine,  or,  after 
drying,  in  Canada  balsam. 

METHODS  OP  STAINING  SPORES. 

The  spores  of  bacteria  were  first  observed  and  de- 
scribed, but  not  rightly  understood,  by  Perty.*  Then 
Pasteur  f  made  a  sharp  distinction  between  the  biol- 
ogy of  an  organism  and  its  spore  without  quite  solv- 
ing the  question  morphologically.  CohnJ  was  the 
first  to  describe  biologically  and  morphologically  the 
formation  and  germination  of  spores.  Further  pecul- 
iarities were  observed  by  Koch,  Brefeld,  Buchner, 
and  especially  Prazmowski,*  who  clearly  described 
the  different  forms  of  the  germination  of  spores  (Fig. 
4 ;  18  and  19).  By  his  observations  this  process  of 
fructification  derived  a  heightened  significance. 

The  observation  of  the  spores  in  the  unstained  con- 
dition finally  followed  (Fig.  4 ;  5  b,  8,  9, 10, 12).  They 
appear,  especially  in  the  hanging  -  drop,  as  strongly 
refracting  round  or  oval  bodies  either  within  the  less 
refractive  bacteria,  or  free  near  these.  Sometimes 
they  are  situated  near  the  middle,  sometimes  at  the 
end.  The  cells  in  which  they  appear  are  sometimes 
unaltered,  sometimes  peculiarly  swollen.  Since  con- 

*  "Zur  Kenntniss  kleinster  Lebensformen,"  1852.     Taf.  XV,  Fig. 
26,  and  following. 

t  "Etudes  sur  la  maladie  des  vers  &  soie,"  1870,  I,  S.  228. 
J  "Beitrage  zur  Biologie  der  Pflanzen,"  1876,  Bd.  II,  2.  Heft, 
S.  263. 

*  "  Ontersuchungen  fiber  die  Entwicklungsgeschichte  und  Fer- 
mentwirkung  einiger  Bakterien-Arten,"  1880,  und  "  Ueber  den  gene- 
tischen  Zusammenhang  der  Milzbrand  und  Heubakterien."   "Biolog. 
Centralblatt,"  1884,  No.  13. 


BACTERIA  AND   MICROSCOPICAL   TECHNIQUE.  75 

densed  bacteria-protoplasm,  according  to  Prazmow- 
ski,  strongly  refracts  light,  it  is  to  be  concluded  that 
a  body  still  more  strongly  light-refracting  is  to  be  re- 
garded as  a  spore. 

Here  belong,  together  with  the  foregoing  morpho- 
logical changes  which  regularly  appear  under  certain 
biological  conditions  in  spores,  their  great  resistance 
to  chemical  agents,  and  especially  to  high  temperature, 
and  the  fact  that  in  the  use  of  watery  or  dilute  alco- 
holic solutions  they  are  not  stained,  but  appear  as  un- 
stained refracting  spaces  within  the  stained  bacteria. 

An  accidental  observation  showed  how  the  spores 
could  also  be  brought  to  view  stained.  Koch  *  saw, 
in  staining  the  tubercle  bacilli  with  aniline-water- 
methyl-blue,  that  the  spores  of  a  species  of  large  bac- 
teria were  stained  blue  at  the  same  time,  while  the 
bacteria  themselves  were  stained  brown  by  the  subse- 
quent treatment.  Gaffky  was  not  able  to  stain  the 
spores  of  other  bacteria  in  the  same  manner.  On  the 
contrary,  Neisser  succeeded  in  staining  the  spores  red 
and  the  bacilli  blue  when  he  used  warm  aniline-water- 
fuchsin  and  subsequently  stained  with  methyl-blue. 
Bienstock  f  also  used  this  staining.  Further,  Buch- 
nerj  discovered  a  means  for  the  isolated  staining 
of  spores.  Because  the  staining  of  the  living  bac- 
teria was  not  successful,  but  those  killed  by  dry- 
ing and  heating  were  readily  colored,  Buchner 
thought  that  the  reason  the  spores  were  not  stained 
was  because  of  the  greater  resistance  of  the  spore- 
membrane.  He  endeavored,  therefore,  to  destroy  the 
membrane  of  the  spores  of  the  bacillus  subtilis,  and 

*  "Mittheilungen,"  1884,  Bd.  II,  Tafel  V,  Fig.  23. 
t  "Zeitschrift  fur  klin.  Med.,"  1884,  S.  1. 

\  u  Ueber  das  Verhalten  der  Spaltpilzsporen  zu  den  Anilinfar- 
ben."  "  Aerztliches  Intelligenzblatt,"  1884,  No.  33,  S.  370. 


76  BACTERIOLOGICAL  INVESTIGATION. 

thus  make  them  accessible  to  the  staining  fluid.  In 
this  manner  he  succeeded  in  staining  spores  in  dried 
cover-glass  preparations  which  had  been  heated  from 
one  half  to  one  hour  at  a  temperature  of  210°  C.  in  a 
dry-oven,  or  one  hour  in  a  steam-kettle  at  120°  C.  A 
successful  result  was  also  obtained  when  the  prepara- 
tions were  dipped  in  concentrated  English  sulphuric 
acid  for  fifteen  seconds  and  afterward  carefully 
washed,  or  when  they  were  subjected  for  a  longer 
time  to  a  concentrated  solution  of  caustic  soda.  In 
preparations  thus  treated,  especially  in  the  use  of 
methyl-blue,  the  spores  alone  are  stained,  while  the 
bacteria  themselves  no  longer  take  up  color. 

Even  before  the  publication  of  the  brief  article  of 
Buchner,  I  had  endeavored  to  bring  the  spores  into 
view  by  both  isolated  and  double  staining.  Although 
these  investigations  are  not  yet  in  all  directions  con- 
cluded, still  I  shall  now  describe  the  general  results, 
because  this  part  of  the  technique  is  as  yet  little 
known  and  used. 

If  bacteria  are  examined  in  the  hanging -drop 
shortly  before  the  formation  of  spores,  in  many  of 
them  refracting  bodies  are  found  which  have  not 
yet  a  size  equal  to  that  of  spores.  When  the  prepa- 
rations, dried  and  fixed  by  passing  them  through  the 
flame  three  times,  are  stained  with  an  aqueous  or  di- 
lute alcoholic  staining  solution,  the  bacteria  in  this 
stage  are  found  to  be  stained,  not  so  equally  as  before, 
but  some  parts  are  more  deeply  colored.  This  con- 
densed protoplasm  takes  the  dye  more  readily  than 
the  protoplasm  not  thus  concentrated.  (I  had  pre- 
viously noticed  intimations  of  this  in  the  involution 
forms  of  the  bacillus  cyanogenus.)  Then  follows  the 
stage  in  which  the  refracting  corpuscles  become  much 
more  equal  in  size,  but  they  still  stain  well ;  and  finally 


BACTERIA  AND  MICROSCOPICAL    TECHNIQUE.  77 

a  stage  in  which  similar  refracting  corpuscles  are  pres- 
ent in  the  unstained  preparations,  but  these  no  longer 
take  up  the  color.  Now  the  spores  have  first  ob- 
tained an  insusceptibility  to  the  dye  through  the 
formation  of  a  membrane  difficult  of  penetration, 
which  prevents  the  absorption  of  the  coloring-matter. 

In  the  cover-glass  preparations,  which  have  been 
passed  through  the  flame  three  times,  the  bacteria  and 
nuclei  are  stained  well.  If  they  are  passed  through 
the  flame  more  times,  say  six,  the  bacteria  are  stained 
successively  worse,  but  the  nuclei  still  absorb  the  dye 
well,  as  does  also  the  condensed  protoplasm  of  the 
bacteria  which  has  not  yet  formed  spores.  At  this 
stage,  besides  the  nuclei,  granular  elements  can  often 
be  seen  which  may  easily  impress  one  as  belonging 
to  the  badly  stained  bacteria.  If  the  preparations 
are  passed  through  the  flame  still  more  times,  say 
ten,  then  both  the  nuclei  and  the  condensed  proto- 
plasm lose  their  susceptibility  to  the  dye,  and  the 
spores  gain  it. 

In  the  case  of  some  of  the  bacteria  of  putrefac- 
tion, it  is  sufficient  to  pass  the  preparation  through 
the  flame  only  seven  times,  but  in  others  ten  times 
are  required.  (In  the  dry-oven  a  similar  stage  is 
reached  in  from  fifteen  to  thirty  minutes  at  180°  to 
200°  C.)  The  spores  then  take  up  aqueous  solutions 
of  red,  violet,  blue,  brown,  and  green  basic  aniline- 
dyes. 

This  isolated  staining  for  the  proof  of  the  resist- 
ance of  the  spores,  as  Buchner  intended,  is  perhaps 
sometimes  to  be  used ;  but,  since  in  this  way  noth- 
ing is  learned  concerning  the  relation  of  the  spores 
to  the  bacteria,  it  is  better  to  use  a  double  staining. 
The  procedure  is  almost  the  same,  quantitatively  in- 
creased, as  that  for  staining  the  tubercle  bacilli. 


78  BACTERIOLOGICAL  INVESTIGATION. 

Either  the  preparations,  passed  through  the  flame 
three  times,  are  stained  with  a  strong  alkaline  solu- 
tion from  twelve  to  twenty-four  hours  (less  advan- 
tageously by  warming  for  one  hour),  and  afterward 
stained  with  vesuvin,  or  the  aniline- water- dye  solu- 
tions are  used,  of  which  that  of  Neisser,  previously 
described,  has  proved  to  be  the  most  convenient  and 
satisfactory.  Stain  in  hot  aniline-water  f uchsin,  de- 
colorize with  nitric  acid,  then  stain  with  methyl-blue. 
Some  spores  are  stained,  moreover,  by  saturated 
aqueous  or  dilute  alcoholic  solutions,  if  they  are  at 
the  same  time  heated.  The  difference  of  spores,  in 
respect  to  their  susceptibility  to  dyes,  seems  to  be 
scarcely  less  than  that  of  the  bacteria  themselves. 

PKEPAKATION  OF  SECTIONS. 

Pieces  of  fresh  organ  can  be  cut  with  the  freez- 
ing microtome.  The  sections  are  placed  in  a  one  half 
per  cent  solution  of  sodium  chloridum,  and  in  part 
examined  fresh,  in  part  stained.  For  the  latter  pur- 
pose, the  section,  according  to  Weigert,*  is  spread 
out  upon  a  section-lifter  in  the  salt  solution  with  the 
help  of  a  needle,  is  then  taken  out,  and  the  excess  of 
the  fluid  removed  with  filter-paper.  Then  the  section 
is  placed  in  absolute  alcohol,  introducing  it  slowly, 
where  it  remains  at  least  until  the  disappearance  of 
all  the  air-bubbles  arising  from  the  thawing  of  the 
frozen  specimen.  Then  the  section  is  placed  in  a 
staining  solution,  for  which  purpose  vesuvin  is  most 
recommended,  because  for  staining  in  the  other  dyes 
the  sections  must  remain  longer  in  alcohol.  Accord- 
ing to  Friedlander,f  the  section  can  also  be  taken  out 

*  Virchow's  "  Archiv  fur  pathologiache  Anatomic,"  Bd.  LXXXIV, 
1881,  S.  290. 

t  "  Mikroskopische  Technik,"  2.  Aufl.,  S.  119. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.   79 

of  the  salt  solution  and  placed  directly  in  the  brown 
staining  fluid,  then  for  a  short  time  in  alcohol,  and 
finally  in  glycerine,  or  oil  of  cloves  and  Canada  balsam. 

For  the  study  of  bacteria,  it  seems  to  me  that  the 
preparation  of  fresh  sections  is  entirely  superfluous 
work.  In  the  same  time  in  which  available  sections 
for  this  purpose  can  be  made,  a  dozen  dried  cover- 
glass  preparations  of  the  tissue-juice  can  be  obtained, 
which  furnish  better  information  as  to  the  presence 
and  import  of  bacteria. 

For  the  exact  study  of  the  presence  and  distribu- 
tion of  bacteria  in  the  tissues,  it  is  necessary  to  harden 
the  tissues  thoroughly,  and  from  the  hardened  speci- 
mens to  prepare  with  the  microtome  a  series  of  deli- 
cate sections.  These  sections  are  partly  examined  un- 
stained and  partly  after  staining.  Since  both  the 
staining  itself,  as  well  as  the  maximal  decolorization, 
is  dependent  upon  the  condition  of  the  preparation 
produced  by  hardening,  it  is  of  the  first  importance 
for  bacteria  specimens  to  use  absolute  alcohol  as  a 
hardening  agent.  The  susceptibility  to  dyes,  of  tis- 
sues hardened  by  other  agents,  as  chromic-acid  salts, 
is  inconstant ;  but  possibly  these  hardening  agents 
are  for  other  parasitic  micro-organisms  as  valuable 
as  alcohol.  (Cf.  page  68.) 

The  susceptibility  of  the  albuminates  to  dyes  de- 
pends very  much  upon  the  presence  of  water ;  and 
since  the  albumen  coagulated  by  alcohol  retains  a 
certain  amount  of  water,  which  in  the  course  of  a  few 
days  is  entirely  removed  by  the  absolute  alcohol,  the 
hardening  in  alcohol  must  continue  until  this  condi- 
tion is  constant.  For  this  purpose  a  small  piece  of 
the  organ,  about  the  size  of  a  hazel-nut,  is  allowed  to 
remain  at  least  three  days  in  a  large  quantity  of  fre- 
quently changed  absolute  alcohol. 


80  BACTERIOLOGICAL  INVESTIGATION. 

In  unstained  sections  from  fresh  or  hardened  tis- 
sue, the  bacteria  are  made  visible  by  their  resistance 
to  acids  and  alkalies.*  The  sections  are  fully  cleared 
up  by  50  per  cent  acetic  acid  or  1  to  3  per  cent  caustic 
soda  or  potash.  The  bacteria  withstand  the  action  of 
these  reagents,  as  was  shown  by  von  Recklinghausen 
(page  38).  Old  spirit  preparations  are  heated  in  these 
solutions  to  near  the  boiling-point.  In  preparations 
which  have  thus  been  made  transparent,  the  bacteria 
can  be  sometimes  recognized  by  the  characteristic 
forms  of  the  individual  germs,  as  was  found  by 
Baumgarten  (page  38)  for  the  tubercle  bacillus,  and 
by  Friedlander  for  the  typhoid  bacillus.  In  the  case 
of  bacteria  not  distinguishable  by  the  characteristic 
form  of  the  single  individuals,  especially  in  the  cocci, 
their  grouping  as  diplococci,  sarcina,  torula,  or  zo- 
ogtoa  is  often  characteristic.  These  bodies,  of  equal 
size,  withstand  the  action  of  ether  and  chloroform, 
unlike  fat-drops,  which  may  be  confounded  with  them. 
In  the  vessels  the  masses  of  cocci  are  characterized, 
according  to  von  Recklinghausen,  by  the  production 
in  the  course  of  their  growth  of  a  varicose  condition 
of  the  vessels. 

If  we  find,  in  a  section  of  fresh  or  hardened  tissue, 
masses  or  chains  of  small  bodies  which  are  of  a  simi- 
lar size,  and  which  withstand  both  the  action  of  al- 
cohol and  ether,  and  the  treatment  with  concentrated 
acetic  acid  and  the  alkalies  after  warming,  these  are 
to  be  considered,  according  to  Friedlander,  f  as  micro- 
organisms. 

More  important  is  the  determination,  by  staining, 
of  the  presence  of  bacteria  in  sections  hardened  in  al- 

*  Friedlander's  "  Zusammenstellung  in  der  mikroskopischen  Tech- 
nik,"  2.  Aufl.,  8.  45. 

t  "  Mikroskopische  Technik,"  2.  Aufl.,  S.  46. 


BACTERIA  AND  MICROSCOPICAL  TECHNIQUE.    81 

cohol.  The  necessary  duration  of  the  hardening  of 
tissues  in  alcohol,  to  produce  constant  reaction  to 
dyes,  diminishes  the  susceptibility  of  many  elements 
to  the  dyes,  so  that  the  staining  of  the  hardened  sec- 
tions is,  as  a  rule,  a  quantitative  increase  of  the  meth- 
ods described  for  the  dried  cover-glass  preparations. 
The  sections  are  first  overstained,  and  then,  by  a  sec- 
ond maximal  decolorization,  reduced  to  a  proper  de- 
gree of  color  for  the  nuclei  and  bacteria. 

The  sections  which  have  been  cut  in  alcohol  and 
afterward  placed  in  the  same  fluid  are  put  into  a 
saturated  aqueous  or  diluted  alcoholic  solution  of  the 
dye,  in  which  they  should  remain  from  five  to  thirty 
minutes.  By  warming  to  40°  or  50°  C.,  the  time  re- 
quired can  be  shortened  somewhat,  and  often  also  the 
intensity  of  the  staining  be  increased.  Gentian-vio- 
let, according  to  Weigert,  is  used  very  advantageous- 
ly in  a  1  per  cent  aqueous  solution.  The  sections 
are  stained  diffusely  in  this. 

In  this  method  of  staining  the  previously  much- 
used  decolorization  by  acetic  acid  is  omitted,  because 
many  bacteria,  such  as  the  bacillus  of  typhoid  fever 
and  glanders,  give  up  their  color  more  or  less  com- 
pletely in  a  short  time.  The  sections  are  placed  in 
distilled  water  to  remove  an  excess  of  the  color.  They 
are  then  carefully  spread  out  upon  a  section-lifter, 
and  with  this  slowly  dipped  in  absolute  alcohol  for 
differentiation  of  the  bacteria  and  nuclei  and  for  de- 
hydration. They  remain  for  a  few  minutes  in  alco- 
hol, which  must  be  absolutely  free  from  acid,  then 
are  cleared  up  in  oil  of  turpentine  or  cedar,  and  can 
be  immediately  examined  in  this  medium.  For  pre- 
serving, the  oil  is  removed  with  filter-paper,  and  they 
are  then  mounted  in  Canada  balsam.  Since  the  bal- 
sam is  soluble  in  the  immersion-oil,  it  must  be  suf- 


82  BACTERIOLOGICAL  INVESTIGATION. 

ficiently  hard  to  prevent  solution  by  the  shifting  of 
the  cover-glass.  If  it  is  desired  to  examine  or  send 
away  preparations  freshly  mounted  in  balsam,  it  is 
advisable  to  fix  the  cover-glass  upon  the  slide  with 
shellac  or  gold-size,  which  are  not  soluble  in  the  im- 
mersion-oil. Many  different  dyes  are  also  available 
for  use  in  staining  sections. 

In  the  use  of  the  method  of  staining  just  de- 
scribed, the  typhoid  bacillus  stains  badly  ;  the  spiril- 
lum of  relapsing  fever  only  in  brown,  and  then  not 
well ;  and  the  bacillus  of  leprosy  badly  in  brown,  but 
well  in  red  and  blue  dyes.  For  these  cases  the 
greatest  intensity  is  obtained  by  the  use  of  the  strong 
alkaline  solution  (page  49),  which,  on  this  account, 
according  to  Loffler,  up  to  the  present  time  has 
proved  the  most  universally  valuable  solution  for 
sections. 

The  sections  are  placed  in  this  solution  for  a  few 
minutes,  then  for  a  few  seconds  in  £  to  1  per  cent 
acetic  acid,  and  moved  to  and  fro  in  order  to  remove 
the  excess  of  color  from  the  tissue  and  to  differenti- 
ate the  bacteria  and  nuclei ;  then  are  dehydrated  in 
alcohol,  cleared  up  in  oil  of  cedar,  and  preserved  in 
Canada  balsam.  By  this  treatment  many  forms  of 
bacteria,  otherwise  difficult  to  stain,  are  well  stained ; 
the  tubercle  bacillus  as  well  as  by  other  methods,  also 
the  bacillus  of  glanders  (which  in  the  aniline- water 
solutions  is  decolorized  by  the  acetic  acid) ;  the  ty- 
phoid bacillus  and  the  spirillum  of  relapsing  fever, 
which  had  been  previously  stained  only  very  defec- 
tively in  any  other  manner.  In  the  case  of  most 
other  bacteria,  no  great  difference  has  been  noticed 
as  to  the  value  of  this  method.  The  comparative 
staining  by  the  use  of  aqueous  and  alkaline  solutions 
sometimes  allows  the  recognition  of  pure  microscop- 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    83 

ical  differences  which  are  available  for  the  differen- 
tial diagnosis  in  morphologically  similar  forms. 

For  the  isolated  staining  of  bacteria  in  sections, 
according  to  Koch,  the  sections  are  brought  from  the 
staining  solution  into  a  solution  of  potassium  carbon- 
ate, which  is  prepared  by  the  mixture  of  equal  parts 
of  distilled  water  and  a  saturated  solution  of  the  salt. 
The  sections  remain  in  this  solution  for  about  five 
minutes,  and  then  are  transferred  with  a  section- 
lifter  to  alcohol,  cleared  up  in  oil  of  cedar,  and  pre- 
served in  balsam.  In  this  method  different  dyes  can 
be  used.  If  the  sections  are  stained  in  the  aniline- 
oil-gentian-violet  solution,  the  isolation,  according  to 
the  Gram  method  (page  60),  is  still  more  beautiful  for 
most  bacteria.  The  sections  are  taken  from  the  al- 
cohol in  which  they  are  placed  after  cutting,  and  put 
from  one  to  three  minutes  (tubercle  bacilli  in  sections 
twelve  to  twenty-four  hours)  in  an  aniline- water  solu- 
tion of  gentian- violet.  Then,  either  without  washing 
or  after  gently  washing  in  alcohol,  they  are  trans- 
ferred to  the  iodine  and  potassium  iodide  solution,  in 
which  they  remain  one  to  three  minutes.  In  the 
iodine  solution  a  precipitation  occurs,  and  the  sec- 
tions are  stained  a  blackish  purple  ;  they  are  then 
placed  in  alcohol  until  completely  decolorized,  cleared 
up  in  oil,  and  preserved  in  balsam  as  usual.  The 
bacteria  appear  dark  blue,  the  nuclei  and  tissue  pale 
yellow.  The  capsule-cocci  of  pneumonia  (at  least  as 
a  rule),  and  the  typhoid  bacilli  are  decolorized  like 
the  nuclei. 

Double  staining  can  be  obtained  in  sections  which 
have  been  treated,  according  to  the  Gram  method,  by 
placing  them  in  an  aqueous  solution  of  vesuvin  after 
the  decolorization  in  alcohol.  They  are  then  dehy- 
drated in  alcohol,  cleared  up  in  oil,  and  mounted  in 


84:  BACTERIOLOGICAL  INVESTIGATION. 

balsam.  The  nuclei  are  stained  brown,  while  the 
bacteria  remain  blue. 

As  already  stated,  carmine  and  hsematoxylin  do 
not  stain  all  bacteria,  and  even  those  stained  are  not 
as  well  stained  as  by  the  basic  aniline-dyes  ;  but  they 
are  nuclei-staining  agents  of  the  first  order.  Sec- 
tions in  which  the  bacteria  are  stained  blue,  accord- 
ing to  the  Koch  method,  can  afterward  be  placed  in 
a  solution  of  carmine  or  hsematoxylin  for  about  ten 
minutes,  in  order  to  stain  the  nuclei.  They  are  then 
likewise  treated  with  alcohol,  oil,  and  balsam. 

If  the  sections  are  diffusely  stained  in  concentrated 
aqueous  or  diluted  alcoholic  solutions  of  dyes,  they 
are  first  put  into  alcohol  for  the  differentiation  of  the 
bacteria  and  nuclei ;  then,  for  the  removal  of  the 
alcohol,  for  a  moment  in  water,  and  afterward  into 
a  solution  of  carmine  or  hsematoxylin,  according  as 
the  first  staining  was  with  a  blue  or  red  aniline-dye. 

For  these  cases,  according  to  Weigert,  a  1  per 
cent  solution  of  gentian- violet  and  a  subsequent 
staining  with  picro- carmine  is  especially  recom- 
mended. The  time  required  for  the  ordinary  nuclei 
staining  (about  ten  minutes)  must  here  be  somewhat 
extended,  because  the  carmine  must  displace  the  gen- 
tian-violet from  the  nuclei.  The  time  required  is  from 
a  half -hour  to  an  hour.  By  a  still  longer  action  the 
carmine  is  also  partially  substituted  for  the  aniline- 
dye  in  cocci. 

These  different  elective  affinities  of  the  bacteria 
and  nuclei  for  dyes  can  be  used,  perhaps,  yet  more 
advantageously  if  the  sections  are  first  placed  for  ten 
or  fifteen  minutes  in  a  solution  of  carmine  or  hsema- 
toxylin. In  this  time  a  good  and  durable  staining  of 
the  nuclei  is  obtained,  while  the  bacteria  are  either 
unstained  or  badly  stained.  After  washing  in  water, 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    85 

the  sections  are  then  transferred  to  a  solution  of  one 
of  the  basic  aniline-dyes  in  which  the  bacteria  are 
stained,  while  the  first  dye-stuff  is  not  displaced  from 
the  nuclei.  They  are  then  treated  as  usual  with  alco- 
hol, oil,  and  balsam.  If  picro-carmine  is  used  in  place 
of  the  ordinary  carmine,  a  threefold  staining  is  ob- 
tained (page  51). 

The  so-called  plasma-cells  in  sections  can  be  easily 
confounded  with  cocci.  They  are  spherical  or  spin- 
dle-shaped cells  with  a  coarsely  granular  protoplasm, 
the  granules  of  which  react  to  the  basic  aniline-dyes 
in  the  same  manner  as  most  cocci.  The  nuclei  of 
these  cells  are  not  stained,  so  that  the  appearance  of 
colonies  of  cocci  is  closely  simulated,  with  which  they 
have  often  been  confounded. 

These  granules  do  not  show  the  same  resistance  to 
acids  and  alkalies,  and  are  not  of  quite  such  equal 
size  as  the  cocci.  Further,  they  are  especially  to  be 
recognized  by  their  position,  as  they  are  commonly 
found  on  the  walls  of  the  vessels.  A  careful  study  of 
these  cells  is  in  the  highest  degree  essential,  and  the 
ear  of  the  white  mouse  is  especially  recommended  for 
this  purpose.  An  exact  differentiation  is  often  only 
to  be  obtained  by  the  isolated  staining  of  the  bacteria, 
in  which  the  plasma-cell  granules  remain  unstained. 

For  the  simultaneous  staining  of  most  cocci  and 
the  plasma-cells,  the  following  solution  of  Ehrlich  and 
Westphal  is  recommended,  which  also  permits  of  the 
differentiation  of  other  bacteria  from  the  nuclei : 
Partsch-Grenacher' s  carmine  (carmine  pur.,  prts.  2; 
aq.,  200 ;  alum,  5 ;  boil  for  fifteen  minutes  ;  fil- 
ter ;  add  carbolic  acid,  1  part) 100  c.  cm. 

Glycerine 100  c.  cm. 

Cone,  alcoholic  sol.  dahlia 100  c.  cm. 

Gflacial  acetic  acid. .  20  c.  cm. 


86  BACTERIOLOGICAL  INVESTIGATION. 

In  this  solution  the  sections  remain  twenty-four 
hours,  then  are  transferred  for  a  few  minutes  to  alco- 
hol, afterward  treated  with  oil  and  balsam.  In  this 
solution  the  nuclei  take  a  red,  the  plasma-cell  gran- 
ules and  bacteria  a  blue  or  violet  color. 

For  the  demonstration  of  the  capsule  cocci  of 
pneumonia,  Friedlander  now  uses  : 

Cone,  alcoholic  solution  gentian-violet 50  c.  cm. 

Aq.  destil 100  c.  cm. 

Ac.  acetic 10  c.  cm. 

The  sections  remain  in  this  solution  twenty-four 
hours,  and  then  are  placed  for  differential  decoloriza- 
tion  in  1  per  cent  acetic  acid  for  a  few  minutes ;  after- 
ward, as  usual,  in  alcohol,  oil,  and  balsam. 

A  contingent  differential  diagnosis  is  possible  mi- 
croscopically from  the  fact  that  by  the  use  of  the 
Gram  method  these  capsule  cocci  are  decolorized, 
while  the  remaining  cocci  all  (?)  retain  the  color. 

Babes*  recommends  safranin  for  the  staining  of 
bacteria  in  sections.  The  sections  are  allowed  to  re- 
main a  half -hour  in  a  mixture  composed  of  equal  parts 
of  a  concentrated  aqueous  and  a  concentrated  alcoholic 
solution  of  the  dye.  Then  they  are  put  for  a  short 
time  in  water,  and  a  few  minutes  in  alcohol ;  after- 
ward in  oil  of  turpentine  and  balsam.  The  bacteria 
are  stained  red,  and  sometimes  have  almost  an  iso- 
lated staining.  This  method  offers  no  advantage, 
since  the  cocci  are  well  stained,  but  other  bacteria 
partly  not  at  all  and  partly  much  worse  than  by 
other  methods. 

The  typhoid  bacilli  are  ordinarily  more  difficult 
to  stain  than  most  other  bacteria,  even  if  the  solution 
is  warmed.  According  to  Gaffky,f  it  is  best  to  allow 

*  "  Archiv  f.  mikroskopische  Anatomie,"  Bd.  XXII,  S.  359. 
t  "Mittheilungen,"  1884,  Bd.  II,  S.  378. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    87 

the  sections  to  remain  from  twenty  to  twenty-four 
hours  in  a  deep-blue  opaque  solution,  which  is  pre- 
pared fresh  each  time  by  the  addition  of  a  saturated 
alcoholic  solution  of  methyl-blue  to  distilled  water. 
They  are  then  washed  in  distilled  water  entirely  free 
from  acid,  dehydrated  in  absolute  alcohol,  cleared 
up  in  oil  of  turpentine,  and  preserved  in  balsam. 
They  are  also  well  stained  in  the  alkaline  solution  of 
methyl-blue,  while  by  the  use  of  the  Gram  method 
they  are  decolorized. 

The  bacilli  of  glanders  are  decolorized,  after  stain- 
ing in  an  aniline  aqueous  solution,  by  water  contain- 
ing acetic  acid.  However,  they  are  well  stained  in  an 
alkaline  solution  of  methyl-blue. 

The  bacilli  of  leprosy  in  sections  are  stained  the 
same  as  tubercle  bacilli.  For  differential  diagnosis 
Baumgarten  recommends  the  following  (page  65) : 
The  sections  are  placed  for  twelve,  or  at  most  fifteen, 
minutes  in  a  dilute  alcoholic  solution  of  fuchsin, 
then  thirty  seconds  in  acid  alcohol  (nitric  acid  one 
part,  alcohol  ten),  to  decolorize  them  ;  washed  in  dis- 
tilled water,  dehydrated  in  alcohol,  then  treated  with 
oil  and  balsam.  In  this  time  the  bacilli  of  leprosy 
are  well  stained,  while  the  tubercle  bacilli  are  not 
stained  at  all. 

The  tubercle  bacilli  can  be  brought  out  if  the  sec- 
tions are  placed  for  twelve  hours  in  a  weak  alkaline, 
or  for  one  hour  in  a  strong  alkaline  solution  of  methyl- 
blue  (page  49),  then  for  a  few  minutes  in  a  concen- 
trated aqueous  solution  of  vesuvin,  and  finally  in  al- 
cohol. They  can  also  be  made  visible  by  the  Gram 
method.  For  the  differential  diagnosis  the  sections 
are  stained  with  the  greatest  certainty,  according  to 
the  previously  described  principle,  in  the  aniline- 
water-dye  solutions  of  Ehrlich  or  Weigert  -  Koch. 


88  BACTERIOLOGICAL  INVESTIGATION. 

The  sections  remain  twelve  to  twenty-four  hours  in 
an  aniline-water,  methyl-violet,  or  fuchsin  solution, 
then  a  few  minutes  in  dilute  nitric  acid  (1  to  3  or  4) ; 
are  washed  in  60  per  cent  alcohol  for  a  few  minutes, 
stained  in  a  dilute  aqueous  solution  of  vesuvin  or 
methyl-blue,  washed  in  60  per  cent  alcohol,  dehy- 
drated in  absolute  alcohol,  cleared  up  in  oil  of  cedar, 
and  finally  imbedded  in  Canada  balsam. 

The  vibriones  of  cholera  Asiatica  can  be  stained  in 
sections  like  most  bacilli. 

The  spirilla  of  relapsing  fever  are  stained  in  aque- 
ous and  glycerine  solutions  of  vesuvin,  but  not  well 
in  other  dyes.  On  the  other  hand,  they  are  stained 
well  in  strong  alkaline  solutions  of  methyl-blue. 

A  few  remarks  are  still  necessary  concerning  the 
epiphytic  bacteria  and  the  other  epiphytic  micro- 
organisms similar  to  them.  (Whether  the  parasitic 
bacteria  are  to  be  classed  with  the  epiphytic  has  not 
yet  been  certainly  determined.)  The  chief  difficulty 
in  this  investigation  is  due  to  the  presence  of  fat. 
Balzer*  washed  the  scales  of  the  epidermis  with 
ether  and  alcohol,  and  examined  them  after  staining 
with  an  alcoholic  solution  of  eosin,  or  without  pre- 
vious staining,  in  a  40  per  cent  solution  of  caustic 
soda.  According  to  von  Sehlen,f  the  hairs,  after  the 
removal  of  the  fat,  are  placed  for  a  short  time  in 
alcohol,  and  then  in  a  dilute  aniline- oil  solution  of 
fuchsin.  They  are  afterward  washed  in  acid  alcohol 
(concentrated  hydrochloric  acid  1  part,  75  per  cent 
alcohol  99  parts) ;  the  excess  of  the  acid  is  removed 
with  distilled  water,  and  a  double  staining  is  pro- 

*  "  Contribution  &  I'Stude  de  l'6ryt&me  tricophytique."  "Arch. 
de  physiol.,"  3.  s6r.,  1883,  Bd.  I,  S.  171. 

f  "  Mikrokokken  bei  Area  Cilei."  "  Fortschritte  der  Medizin," 
1883,  No.  23. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.    89 

duced  by  the  use  of  a  concentrated  aqueous  solution 
of  gentian-violet,  and  then  alcohol  is  used  for  differ- 
entiation as  before. 

According  to  Bizzozero,*  the  epidermis  is  touched 
with  a  cover-glass,  which  is  afterward  passed  through 
a  flame  three  times.  The  fat  is  removed  by  chloro- 
form, and  the  preparation  is  stained  by  fuchsin  or 
gentian-violet.  For  the  removal  of  fat  from  the  epi- 
dermic scales,  they  are  placed  for  a  few  seconds  in 
alcohol,  then  for  a  day  or  two  in  ether,  and  afterward 
again  in  alcohol.  After  the  fat  has  been  removed 
there  are  three  methods  of  procedure :  1.  A  drop,  con- 
sisting of  equal  parts  of  water  and  acetic  acid,  or  of  a 
10  per  cent  solution  of  caustic  potash,  is  placed  upon 
a  slide.  The  epidermic  scales  from  which  the  fat  has 
been  removed  are  placed  in  this  drop  and  allowed  to 
swell,  then  a  cover-glass  is  laid  upon  them.  For  the 
preservation  of  these  preparations,  glycerine  is  intro- 
duced from  the  edge. 

2.  The  epidermic  scale  is  spread  out  with  a  needle 
in  glycerine,  slightly  tinged  with  methyl-blue.     In 
this  the  fungi  are  stained  blue,  while  the  epidermic 
cells  remain  unstained. 

3.  A  drop  of  50  per  cent  acetic  acid  is  put  upon 
a  cover-glass,  and  the  epidermic  scales,  with  the  fat 
removed,  are  placed  in  it.     After  fifteen  minutes  or 
more,  if  the  scales  are  well  swollen,  they  are  spread 
out  with  a  needle ;   then  the  acetic  acid  is  evapo- 
rated by  gentle  heat.     (For  this  purpose,  the  cover- 
glass  may  be  held  high  over  a  flame  and  moved  to 
and  fro.)    After  drying,  the  preparation  is  passed 
through  the  flame  three  times,  and  then  stained  for 
ten  to  thirty  minutes  by  adding  a  drop  of  an  aque- 

*  "  Ueber  die  Mikrophyten  der  normalen  Oberhaut  des  Menschen." 
Virchow's  «  Archiv,"  1884,  Bd.  XCV1II,  S.  441. 


90  BACTERIOLOGICAL  INVESTIGATION. 

eras  solution  of  methyl- violet,  gentian-violet,  vesuvin, 
methyl-blue,  or  of  a  dilute  alcoholic  solution  of  fuch- 
sin.  It  is  then  carefully  washed,  dried,  and  preserved 
in  balsam.  Methyl-blue  is  preferred  by  Bizzozero, 
because  only  the  fungous  elements  are  stained  with 
it.  I  have  used  most  advantageously  the  strong  alka- 
line solution  of  methyl-blue,  allowing  it  to  act  for 
about  five  minutes.  With  this  slight  modification,  it 
seems  to  me  that  this  third  method  of  Bizzozero  is 
at  present  most  serviceable  for  the  determination  of 
bacteria. 

Presently  I  will  show  that  in  true  mycosis  the 
mycelial  filaments  of  the  mold  fungus  are  best 
brought  out  by  the  method  of  Loffler  (page  82). 
The  presence  of  radiating  fungi  in  tissue  -  sections 
can  generally  be  determined  without  special  prepara- 
tion. But  in  the  frequent  calcification  of  the  glands 
in  actinomycosis  it  is  often  necessary  to  first  decal- 
cify, by  hardening  them  in  alcohol  containing  hydro- 
chloric acid,  and  then  to  place  them  in  absolute  alcohol. 
The  staining  of  Weigert  *  is  also  useful.  According 
to  this  the  sections  are  placed  for  one  hour  in  a  solu- 
tion of  orchilla  (rock  moss).  Pure  orchilla,  which  has 
previously  lain  for  a  long  time  in  the  air  to  allow  the 
escape  of  the  ammonia,  is  dissolved,  according  to 
Wedl,  in  such  quantities,  in  a  mixture  of  20  c.  cm. 
absolute  alcohol,  5  c.  cm.  acetic  acid,  and  40  c.  cm.  of 
distilled  water,  that  the  fluid  becomes  dark  red,  and 
after  filtering  appears  ruby-red.  Then  the  section  is 
washed  in  alcohol,  placed  in  a  1  per  cent  solution  of 
gentian-violet  and  treated  as  for  the  staining  of  bac- 
teria. In  these  sections  the  nuclei  are  stained  blue- 
violet,  the  connective  tissue  a  pale  orange,  the  inte- 
rior of  the  refracting  fungi  a  pale  blue,  and  the  outer 
*  Virchow's  "  Archiv,"  1881,  Bd.  LXXXIV,  S.  245. 


BACTERIA  AND  MICROSCOPICAL   TECHNIQUE.   91 

parts  a  ruby-red,  often  separated  by  a  colorless  zone 
from  the  central  portion. 

For  free  amoeba  and  cells  without  membranes 
(page  68)  Brass*  recommends  a  solution  of  1  part 
chromic  acid,  1  part  platinum  chloride,  1  part  cone, 
acetic  acid,  and  400  to  1,000  parts  of  water.  For  hard- 
ening the  tissues,  in  which  the  cells  ought  to  be  as 
little  altered  as  possible  (vide  page.  79),  Brass  recom- 
mends a  i  to  J  per  cent  solution  of  chromic  acid,  to 
which  a  few  drops  of  a  concentrated  solution  of  the 
same  salt  is  added  later.  After  some  time  the  prepa- 
ration is  placed  in  30  per  cent  alcohol,  and  generally, 
for  the  complete  removal  of  the  water,  in  alcohol 
gradually  made  stronger,  and  finally  in  absolute  al- 
cohol. The  mixture  of  chromic  acid,  platinum  chlo- 
ride, and  acetic  acid  also  produces  excellent  harden- 
ing, especially  if  four  to  six  drops  of  1  per  cent  osmic 
acid  is  added  to  100  grammes  of  the  solution.  The 
staining  is  effected  by  borax,  or  ammonio-carmine,  or 
a  solution  of  hsematoxylin. 

*  "  Die  Methoden  bei  der  Untersuchung  thierischer  Zellen."  "  Zeit- 
schrift  f.  wissenschaftl.  Mikroskopie,"  1884,  S.  39. 


III. 

CULTURE-METHODS;    PURE  CULTURES. 

EHRENBERG  first,  and  later  Cohn  and  Schroder, 
expressed  the  belief  that  there  were  true  species 
among  bacteria.  On  the  grounds  of  the  investigations 
of  Cagniard-Latour  and  Schwann,  Turpin  reached 
the  conclusion  concerning  fermentation,  and  Henle 
concerning  the  infectious  diseases,  that  specific  de- 
compositions and  diseases  are  produced  by  specific 
micro-organisms ;  and  Pasteur,  by  inoculation  experi- 
ments, proved  experimentally  that  specific  and  differ- 
ent micro-organisms  underlie  specific  decompositions. 
These  views,  as  well  as  those  of  their  opponents,  who 
argue  for  inconstancy  of  form  and  action  among 
micro-organisms,  can  only  be  conclusively  proved 
when  they  are  observed  pure  and  free  from  all  ad- 
mixtures. For  this  reason  it  has  been  the  endeavor 
of  many  investigators,  for  a  long  time,  to  establish 
methods  of  obtaining  pure  culture  of  bacteria,  which 
would  be  free  from  all  objections. 

1.  TRANSPARENT  FLUID  CULTURE-MEDIA. 

This  method  is  the  oldest,  and  underlies  all  the 
experiments  of  the  earlier  time  on  fermentation.  It 
was  used  for  the  cultivation  of  bacteria  by  Pasteur  * 

*  "Memoire  sur  la  fermentation  appellee  lactique."  "Compt. 
rend.,"  1857,  Bd.  XLV,  S.  913. 


CULTURE-METHODS;  PURE  CULTURES.          93 

in  his  first  work  on  the  vital  fermentation  theory. 
The  principle  of  this  method,  consists  in  transferring 
a  particle  of  the  original  fluid  containing  the  micro- 
organisms of  fermentation,  or  a  particle  of  yeast 
(which  contains  them  in  itself),  to  a  fluid  compounded 
to  resemble  as  nearly  as  possible  the  original  one ; 
from  this  again  a  particle  is  transferred  to  a  third 
solution,  etc.  The  school  of  Pasteur  has  adhered 
almost  exclusively  to  the  use  of  the  transparent  fluid 
media,  even  up  to  the  present  time,  often  in  its  origi- 
nal form  ;  also  often  with  its  modification,  the  meth- 
od of  dilution,  later  to  be  described. 

The  important  morphological  investigations  of 
Cohn  were  also  joined  closely  to  this  method. 

In  fluids  which  undergo  decomposition  through 
the  action  of  bacteria,  sometimes  a  diffuse  cloudiness 
appears  ;  sometimes  precipitation  occurs  ;  more  often 
a  mouldy  scum  is  formed.  In  transfer  experiments  a 
particle  is  transferred  to  a  second  solution  from  each 
of  the  portions  appearing  differently.  This  transfer- 
rence  to  a  new  solution,  briefly  spoken  of  as  inocu- 
lation, is  performed  as  follows :  a  platinum-needle, 
previously  heated  and  cooled,  straight  or  bent  into  a 
loop,  is  brought  in  contact  with  the  mycoderma  (the 
mouldy  scum),  or  is  dipped  into  the  cloudy  fluid,  and 
then  the  particle,  thus  taken  up,  is  introduced  into 
the  new  solution.  In  place  of  the  platinum -needle, 
a  drawn-out  capillary- tube  can  be  used,  with  which 
a  drop  of  the  fluid  is  transferred.  Such  a  particle  or 
drop  consists  chiefly  of  a  great  mass  of  bacteria. 

Following  this  inoculation,  that  species  develops 
first  which  finds  in  the  new  solution  the  best  condi- 
tions for  its  existence.  When  the  culture-material 
is  exhausted  for  one  species,  one  or  another  of  the 
forms,  that  at  first  were  suppressed,  may  develop, 


94  BACTERIOLOGICAL  INVESTIGATION. 

provided  the  particle  or  drop  transferred  contained 
several  species.  It  is  observed,  in  the  course  of  these 
experiments,  that  small  differences  in  conditions,  such 
as  placing  the  inoculated  solution  at  a  lower  or  higher 
temperature,  favor  the  development  of  certain  germs. 

Finally,  following  out  the  conditions  favorable  to 
the  development  of  a  single  germ,  a  more  or  less  pure 
culture  of  this  organism  is  obtained  in  some  one  of 
the  transfers.  But  this  is  not  always,  in  fact  it  is 
seldom,  the  organism  which  it  is  desired  to  isolate. 
Ordinarily,  the  result  is  a  pure  culture  of  one  of  the 
common  septic  species  which,  under  the  chosen  con- 
ditions, supplanted  the  other  more  susceptible  forms. 
On  account  of  this  it  happened  that,  in  the  use  of  this 
method  of  culture,  the  bacterium  termo  and  bacillus 
subtilis  often  appeared.  These  play  a  much  greater 
part  in  the  older  bacteria  literature  than  now. 

In  this  way  the  dependence  of  the  growth  of  bac- 
teria on  the  culture-media  was  learned.  This  led  to 
the  choice  of  such  fluids  as  would  offer  approximately 
equally  favorable  conditions  for  development  to  the 
largest  possible  number  of  micro-organisms. 

Pasteur's*  fluid  is  the  oldest  of  these  artificial 
media  for  the  cultivation  of  bacteria.  This  consists 
of  one  part  tartrate  of  ammonia,  ten  parts  sugar,  and 
the  ashes  of  one  part  of  yeast,  to  one  hundred  parts 
of  water. 

A.  Mayer  f  used  in  place  of  the  yeast-ash  a  solu- 
tion of  the  salt  contained  in  it.  Then  Cohn,^:  after 
he  had  used  the  culture-fluid  of  Mayer  with  mineral 

*  "  Annales  de  Cbimie  et  de  Physique,"  Bd.  LVIII,  S.  323.  Deutsch 
von  Griessmayer,  "Die  Alkohol-Gahrnng,"  1878. 

t  "  Unters.  fiber  die  Alkohol-Gahrung,"  1869.  "  Lehrbuch  der 
Gfihrungs-Chemie,"  3.  Aufl.,  1879. 

\  "Beitrage  zur  Biologie  der  Pflanzen,"  I,  2.  Heft,  S.  195. 


CULTURE-METHODS;   PURE  CULTURES.          95 

nutrient  salt,  omitting  the  sugar,  devised  the  follow- 
ing normal  culture-fluid : 

Calcii  phosphati *5  gramme. 

Magnesii  sulph.  (crys.). . .        *5       " 

Calcii  phosp.  (tribas) '05     " 

Aq.  destil 100  c.  cm. 

In  this  1  gramme  ammon.  tartrate  was  dissolved. 
Naegeli  *  discovered  that  for  the  lower  fungi  and 
the  fission  fungi,  nitrogen  can  be  best  assimilated  if  it 
is  present  as  NHa,  not  as  well  as  NH,  still  not  as  well 
as  NO,  and  not  at  all  if  it  is  in  combination  with  other 
elements,  such  as  hydrogen  and  oxygen,  so  that  a  de- 
scending scale  may  be  constructed  from  the  soluble 
albuminates,  to  ammonia  and  nitric  acid.  For  car- 
bon he  arranged  the  following  scale : 

1.  The  forms  of  sugar. 

2.  Mannite ;  glycerine ;  the  carbon  group  in  leu- 
cin. 

3.  Tartaric  acid  ;   citric  acid  ;  succinic  acid ;  the 
carbon  group  in  asparagin. 

4.  Acetic  acid  ;  ethyl-alcohol ;  quinic  acid. 

5.  Benzoic  acid  ;  salicylic  acid ;  the  carbon  group 
in  propylamin. 

6.  The  carbon  group  in  methylamin  ;  phenol. 
Naegeli  established  the  following  descending  scale 

for  the  capacity  of  assimilation  of  nitrogen  and  carbon 
from  their  compounds : 

1.  Albumen  (peptone)  and  sugar. 

2.  Leucin  and  sugar. 

3.  Tartrate  of  ammonia,  or  sal.  ammon.,  and  sugar. 

4.  Albumen  (peptone). 

5.  Leucin. 

*  "  Ernahrung  der  niederen  Pilze  durch  Kohlenstoff-  und  Stick- 
stoflVerbindungen."  "  Untersuchungen  Hber  niedere  Pilze,"  1882, 
S.  1. 


96  BACTERIOLOGICAL  INVESTIGATION. 

6.  Tartrate  of  ammonia ;   succinic  ammonia ;  as- 
paragin. 

7.  Acetate  of  ammonia. 

In  regard  to  1,  it  is  to  be  noted  that  the  bacteria 
must  be  able  to  transform  the  albumen  into  peptone, 
and  to  hydrate  the  milk  and  cane-sugar,  so  that  pep- 
tone and  grape-sugar  are  used  to  the  best  advan- 
tage. 

Naegeli  recommends  as  the  best  for  the  mineral 
elements— 

Potassii  phos 1      gramme. 

Magnesii  sulph '02       " 

Calcii  chlor '01       " 

Fluid 100  " 

If  the  reaction  is  to  be  acid,  Naegeli  uses  acid 
potassi  phos. ;  for  neutral  and  alkaline  solution,  po- 
tassii  biphos.  The  poorer  the  nutrient  elements  of 
the  N  and  C  group  are,  the  less  concentrated  the  salt 
solution  should  be,  while,  with  good  nutrient  elements 
of  the  N  and  C  group,  this  normal  degree  of  concen- 
tration may  be  exceeded.  From  this  Naegeli  devised 
the  following  normal  fluids  for  fission  fungi  : 

1.  Aqua 100  c.  cm. 

Ammon.  tart 1     gramme. 

Potassii  biphos '\       " 

Magnes.  sulph '02     " 

Calcii  chlor 01     " 

2.  Aqua 100  c.  cm. 

Albumen-peptone 1     gramme. 

Potassii  biphos '2       " 

Magnes.  sulph '04     " 

Calcii  chlor *02     " 

3.  Aqua 100  c.  cm. 

Cane-sugar 3     grammes. 

Ammon.  tart • 1     gramme. 


CULTURE-METHODS;  PURE  CULTURES.          97 

Potassii  biphos *2    gramme. 

Magnes.  sulph *04       " 

Calciichlor -02       " 

In  place  of  the  tartrate  of  ammonia,  in  the  3d  for- 
mula, an  equal  amount  of  another  of  the  ammonia 
salts  may  be  used,  or  *5  gramme  of  ammon.  nitrat., 
or  *7  gramme  of  asparagin,  or  *4  gramme  of  urea. 

In  place  of  the  nutrient  salts,  one  tenth  per  cent 
beef-extract  may  often  be  more  conveniently  used. 
For  the  fermentation-experiments,  according  to  Fitz,* 
solutions  may  be  employed  which  contain  three  per 
cent  sugar,  glycerine,  or  mannite,  etc.,  and  one  tenth 
per  cent  beef-extract,  to  which  a  small  quantity  of 
calcii  carb.  has  been  added. 

According  to  previous  experiments  with  cultures 
in  fluids,  as  was  also  the  case  in  the  experiments  of 
Fitz,  it  is  well  to  choose  the  culture-fluid  (normal 
fluid)  with  special  reference  to  the  individual  case. 
In  the  lack  of  other  data  at  the  beginning,  that  fluid 
should  be  selected  (as  was  the  case  in  the  first  experi- 
ments of  Pasteur)  in  which  the  bacteria  are  observed 
to  grow  spontaneously.  For  micro-organisms  which 
are  observed  on  solid  substances,  a  decoction  or  in- 
fusion of  this  substance  is  prepared  :  fresh  soil,  sweet 
dried  fruit,  hay,  roots,  etc.  "A  nutrient  fluid  which 
holds  in  solution  such  substances  as  in  a  solid  state 
furnish  naturally  a  pabulum  upon  which  a  fungus 
develops,"  according  to  Brefeld,t  uin  all  probability 
will  also  form  a  suitable  fluid  for  the  development  of 
this  fungus." 

Solutions  for  fungi  are  kept,  as  a  rule,  slightly 

*uTJeber  Spaltpilzgahrung,"  VII.  "Berichte  der  deutschen 
chemischen  Gesellschaft,"  1882,  XV,  S.  867. 

t  "  Kulturmethoden  zur  Untersuchnng  der  Pilze."  "  Botanische 
Untersuchungen  uber  Schimmelpilze,"  Heft  IV,  1881,  S.  5 


98  BACTERIOLOGICAL  INVESTIGATION-. 

acid;  for  bacteria  they  are  rendered  neutral  or 
slightly  alkaline  by  the  addition  of  ammonia  or  car- 
bonate of  soda,  and  then  are  boiled  and  filtered. 

"In  the  use  of  a  clear  nutrient  fluid  thoroughly 
sterilized,  in  which  the  examination  of  the  fungus  by 
direct  observation  is  made  with  the  same  ease  as  if 
they  lived  in  clear  water,  the  mycological  investiga- 
tion is  turned  into  an  algological — i.  e.,  those  condi- 
tions are  artificially  prepared  in  the  nutrient  solutions 
for  the  development  of  the  fungus,  under  which  we 
naturally  find  the  algae,  as  they  for  the  most  part  live 
in  water." 

These  words  from  Brefeld  (I.  c.,  page  7)  also  forcibly 
point  out  the  chief  advantages  of  the  nutrient  solu- 
tions for  the  culture  of  bacteria. 

These  clear,  usually  neutral  reacting,  fluids  must 
be  carefully  sterilized  after  filtration.  For  this  pur- 
pose the  sterilized  cotton  stopper  of  the  sterilized  flask 
or  test-tube  is  removed  with  clean,  previously  heated 
pincettes,  and  laid  upon  its  side  so  that  it  can  not  be 
soiled.  Then,  by  the  aid  of  a  sterilized  funnel  or  pi- 
pette, the  fluid  is  transferred  into  the  flask  until  it  is 
about  half  filled,  or  into  the  test-tube  until  it  is  about 
one  third  filled.  Then  the  stopper  is  again  replaced. 
It  is  often  desirable,  in  addition,  to  cover  the  mouth  of 
the  vessel  with  a  double  layer  of  thick  filter-paper, 
which  is  held  in  place  by  a  rubber  band  and  which 
prevents  the  dust  from  falling  directly  upon  the  cot- 
ton. The  cotton  plug  is  proof  against  the  admission 
of  bacteria,  but  not  always  proof  against  fungi,  as  is 
to  be  observed  when  the  sterilized  vessels  are  pre- 
served for  a  long  time  in  a  moist  chamber. 

The  sterilization  should  be  accomplished  accord- 
ing to  one  of  the  methods  described  in  Chapter  I, 
preferably  in  most  cases  by  the  use  of  the  steam  ster- 


CULTURE-METHODS;  PURE  CULTURES.  99 

ilizing  cylinder.  Small  volumes  of  fluid,  as  those  in 
test-tubes,  are  sterilized  by  an  exposure  for  a  half  to 
three  quarters  of  an  hour,  larger  flasks  in  one  or  two 
hours.  The  time  required  to  bring  the  water  in  the 
cylinder  to  the  boiling-point  should  not  be  included. 
The  test-tubes,  after  being  filled,  are  placed  in  a  wire 
basket  (Fig.  3,  d\  and  then  in  the  steam  cylinder. 

The  manipulations  with  the  fluids  should  be  car- 
ried on  in  a  place  as  free  as  possible  from  micro-or- 
ganisms, in  order  to  prevent  infection  by  air-germs. 
This  may  be  obtained  in  a  laboratory  by  the  prepa- 
ration of  a  glass  case  (similar  to  those  arranged  over 
delicate  balances),  in  which  the  inoculations  and 
transfers  are  made.  The  walls  of  this  case  are  rubbed 
with  moist  cloths,  and  the  air  is  kept  moist  by  vessels 
of  warm  water. 

The  inoculations  are  made  by  a  platinum-needle, 
straight  or  looped,  which  is  previously  heated  in  a 
flame  and  again  cooled,  or  by  a  capillary  pipette. 
By  means  of  one  of  these  a  particle  or  drop  of  the 
material  or  fluid  to  be  used  for  inoculation  is  intro- 
duced as  quickly  as  possible  into  the  sterilized  fluid, 
after  removal  of  the  cotton  plug.  This  is  then  imme- 
diately replaced.  If  the  solutions  have  stood  for  a 
long  time  without  the  mouths  of  the  vessels  being  pro- 
tected by  filter-paper,  so  that  germs  of  bacteria  and 
fungi  may  have  collected  upon  the  cotton,  it  is  better 
to  burn  the  upper  layer  of  cotton  in  a  flame  before 
removal  of  the  plug.  A  large  number  of  single  ex- 
periments should  be  made  simultaneously.  The  fur- 
ther inoculations  are  made  in  the  same  manner.  The 
inoculated  tubes  are,  according  to  the  case,  kept  at 
the  temperature  of  the  room  or  exposed  to  a  higher 
temperature.  For  the  higher  temperatures  a  brood- 
or  culture-oven  is  used.  This  is  made  of  metal  with 


100  BACTERIOLOGICAL  INVESTIGATION. 

double  walls  for  the  reception  of  water  (Fig.  8),  and 
is  covered  with  felt  or  asbestos  to  prevent  the  loss  of 

heat ;   or  the  oven  is  made 
with  a  third  wall,  and  the 
outer  chamber  is  filled  with 
a  layer  of  sand.     The  dimen- 
sions vary  according  to  need  ; 
the   form,   quadrangular    or 
cylindrical,  is  of  little  mat- 
ter.     In   the    quadrangular 
the  height  and  breadth  of 
______     ______         the  interior  is  about  25  cm., 

and  the  length  varies  from  50 

to  75  cm.  A  thermometer  (t)  and  therm o-regulator  (r) 
serve  for  the  regulation  of  the  temperature.  These 
last  can  be  dispensed  with  if  a  small  gas-pressure 
regulator  is  interposed  between  the  principal  supply- 
pipe  and  the  culture-oven.  It  is  heated  by  petro- 
leum or  gas  flames,  which  vary  in  number,  accord- 
ing to  the  size  of  the  oven.  These  are  surrounded  by 
glass  chimneys  to  protect  them  from  draughts  of  air. 

2.  FRACTIONAL  CULTURES. 

The  inoculations  of  fluids  with  pathogenic  bacteria 
were  first  made  by  Klebs,*  and  the  particle  or  drop 
containing  bacteria  ("  Bakterientropf  en  "  of  other  au- 
thors) was  designated  by  him  as  a  u  fraction."  Klebs 
proceeded  in  this  manner  (I.  c.,  page  46) :  uHe  intro- 
duced a  recently  drawn-out  and  closed  pointed  cap- 
illary-tube to  the  bottom  of  .  the  fluid  containing 
bacteria,  and  then  broke  off  the  point.  The  tube, 
after  withdrawal,  was  again  sealed,  washed  with  strong 
alcohol,  introduced  into  a  sterilized  culture-fluid,  and 

*  "  Beitrage  zur  Kenntniss  der  Mikrokokken."   "  Archly  fur  ex- 
periinentelle  Pathologie,"  Bd.  I,  1873,  S.  31. 


CULTURE-METHODS;  PURE  CULTURES.        101 

was  then  again  broken.  The  fliiid-mediiim  was  con- 
tained in  a  stoppered  flask  and  was  covered  with  a 
layer  of  oil."  This  procedure  was  often  repeated,  and 
"in  this  manner  it  is  possible  to  eliminate  any  im- 
purities, which  may  be  contained  in  the  original  fluid, 
and  to  obtain  those  organisms  pure  which  are  present 
in  this  in  preponderating  number." 

This  method  has  furnished  no  important  discov- 
eries, notwithstanding  the  skillful  working  and  the 
sharp  emphasis  laid  upon  the  development  of  many 
germs  from  the  one  originally  present  in  preponderat- 
ing numbers.  In  this  complication  of  the  method  of 
Pasteur  it  is  evident  that  the  objection  also  holds 
good  that,  as  a  rule,  after  a  series  of  fractional  cul- 
tivations, the  desired  pathogenic  organism  is  not 
present  pure.  But  instead  of  this  almost  any  form 
may  be  present  which  at  the  beginning  may  have  been 
perhaps  entirely  overlooked  on  account  of  their  small 
number ;  or  else  some  ordinary  species  of  septic  bac- 
teria, introduced  by  infection  from  the  air  or  by 
manipulation,  has  been  obtained  pure,  because,  un- 
der the  chosen  conditions,  it  thrived  better  than  the 
more  susceptible  pathogenic  micro-organisms,  and 
quickly  supplanted  these. 

By  the  methods  thus  far  described  pure  cultures 
may  be  finally  obtained,  without  successfully  ac- 
quiring those  forms  pure  which  it  was  desired  to 
obtain.  These  methods  are  on  this  account  only  now 
to  be  used  when  it  is  desired  to  secure  an  organism 
in  pure  or  quantity-culture,  the  source  and  action  of 
which  are  of  no  importance. 

3.  OPAQUE  SOLID  CULTURE-MEDIA. 

Aside  from  fluids,  bacteria  are  observed  develop- 
ing spontaneously  on  solid  substances.  If,  for  exam- 


102  BACTERIOLOGICAL  INVESTIGATION. 

pie,  a  slice  of  cooked  potato  is  allowed  to  stand  in 
the  air,  different  slimy  masses  are  seen  to  spread 
themselves  out  upon  the  surface  of  the  potato.  Small 
slimy  points  of  different  colors  form  upon  the  sur- 
face, which  for  some  time  are  quite  distinct  from 
each  other,  but  become  confluent  in  their  further 
growth. 

These  observations  were  first  methodically  made 
use  of  by  Schroeder  *  for  the  pure  cultures  of  pig- 
ment bacteria.  A  particle  from  one  of  these  slimy 
points,  while  it  is  yet  quite  isolated,  is  transferred 
with  a  platinum -needle  to  the  middle  of  the  cut  sur- 
face of  a  freshly  cooked  potato,  placed  in  a  moist 
chamber  in  order  to  prevent  infection  from  the  air. 

The  method  of  cultivation  on  potato  was  material- 
ly improved  by  Koch.  The  potatoes  were  first  care- 
fully cleaned  from  the  coarse  dirt  by  scrubbing,  then 
laid  for  one  half  to  one  hour  in  a  one  to  five  per  cent 
solution  of  corrosive  sublimate,  and  finally  washed  in 
water.  The  potatoes,  thus  cleansed,  with  the  germs 
adhering  mostly  destroyed  by  the  sublimate,  are  then 
heated  to  secure  certain  sterilization.  This  is  done 
in  the  steam  cylinder.  After  reaching  the  boiling- 
point,  the  potatoes  remain  at  this  temperature  for 
about  an  hour.  While  the  potatoes  are  cooling,  a 
moist  chamber  is  prepared.  Large  bell-jars,  of  the 
form  seen  in  Fig.  9,  are  cleansed  by  rinsing  in  a  one 

FIG.  9. 


*"Ueber  einige  durch  Bakterien  gebildete  Pjgmente."  Cohn's 
"Beitrage  zur  Biologic  der  Pflanzen,"  Bd.  I,  Heft  II,  1872  (2.  Ab- 
druck,  1881),  S.  109. 


CULTURE-METHODS;  PURE  CULTURES.        103 

per  mille  solution  of  corrosive  sublimate.  Upon  the 
bottom  of  the  jar  a  number  of  layers  of  filter-paper, 
moistened  with  sterilized  water  or  a  solution  of  sub- 
limate, are  placed.  Then  the  potatoes  are  held  in  the 
left  hand,  between  the  thumb  and  index  finger,  cut 
with  a  sterilized  knife,  and  laid  in  a  jar  with  the  cut 
surface  upward.  Before  this  operation  the  hands 
should  be  washed  in  a  one  per  mille  solution  of  sub- 
limate. The  ordinary  kitchen-knife  is  used  for  cut- 
ting, and,  previous  to  use,  is  sterilized  by  heating  in 
the  flame,  and  while  cooling  is  protected  from  the 
dust.  For  each  potato  a  fresh  knife  should  be  used. 

Then,  by  means  of  a  platinum-needle  which  has 
been  sterilized  in  the  flame,  a  particle  from  one  of  the 
slimy  points  which  is  still  entirely  isolated,  a  so- 
called  colony,  is  taken,  and  is  either  placed  near  the 
middle  of  the  section  of  potato,  or  several  lines  of 
inoculation  are  lightly  drawn  entirely  across  the  sur- 
face. In  the  first  case  the  colonies  develop  in  the 
middle  of  the  section,  and  gradually  extend  toward 
the  edge  ;  in  the  other,  more  or  less  isolated  colonies 
develop  along  the  lines  of  inoculation,  which  later 
unite  and  extend  throughout  the  lines.  For  new 
inoculation  only  those  colonies  are  used  which  are 
recognized  as  pure  with  the  naked  eye  or  lens,  and 
from  which  a  particle  has  been  used  to  make  a  cover- 
glass  preparation  for  microscopical  control. 

Some  of  the  cultures  can  be  kept  at  the  tempera- 
ture of  the  room,  and  others  at  a  higher  tempera- 
ture in  a  culture-oven. 

The  great  advantage  of  this  method,  as  compared 
with  the  previously  described  methods  with  trans- 
parent fluid  media,  consists  in  this  :  namely,  that 
each  germ,  whether  it  has  been  intentionally  inocu- 
lated upon  the  surface  of  the  potato,  or  is  the  result 


104  BACTERIOLOGICAL  INVESTIGATION. 

of  air-infection,  is  isolated  at  its  point  of  contact  with 
the  surface,  and  there  develops  into  a  colony,  while 
in  fluids  the  different  germs  are  mingled  together. 

Pure  cultures  may  also  be  obtained  in  the  fluid 
media,  but  that  organism  is  not  always  present  of 
which  pure  cultures  are  desired.  In  the  potato-cul- 
tures it  is  possible  to  transfer  the  desired  micro- 
organism, and  in  this  way  separate  it  from  others, 
and  after  several  transfers  obtain  pure  cultures.  How- 
ever, the  transfers  must  be  made  early,  while  the  small 
colonies  developed  from  a  single  germ  are  yet  entire- 
ly isolated,  and  are  solitary  and  pure,  as  the  result  of 
the  isolation.  The  limitations  of  the  method  depend 
on  the  fact  that  potatoes  do  not  furnish  favorable 
conditions  for  the  existence  of  all  organisms,  and,  on 
this  account,  many  species  that  develop  on  potato  do 
not  grow  sufficiently  to  be  visible.  This  is  important, 
because  in  the  solid  opaque  media  the  naked  eye  or 
the  magnifying-glass  can  alone  be  used. 

The  inoculations  on  potatoes  are  more  useful 
when  it  is  desired  to  determine  whether  pure  cultures 
obtained  elsewhere,  especially  of  pathogenic  micro- 
organisms, possess  the  capacity  of  developing  on 
vegetable  material.  Then  the  sterilized  potatoes  are 
inoculated  in  the  same  way  with  these  pure  cultures. 
The  sections  of  the  potatoes  should  be  kept  in  the 
same  manner  in  moist  jars,  and  subjected  to  different 
degrees  of  temperature ;  or,  as  a  greater  precaution, 
a  potato-section  is  placed  upon  a  small  glass  plate, 
which  is  then  lowered  (by  the  help  of  a  strip  of 
nickel  bent  at  a  right  angle)  to  the  bottom  of  a  cylin- 
drical glass  vessel  about  18  cm.  high  and  6  cm.  in 
diameter.  The  glass  cylinder — previously  closed  by  a 
cotton  plug— the  glass  plate,  and  nickel  strip  should 
be  sterilized  in  a  dry-oven  before  being  used. 


CULTURE-METHODS;  PURE  CULTURES.         105 

In  place  of  the  sections  of  potatoes — which,  on  ac- 
count of  their  yellow  or  white  color,  are  used  in  prefer- 
ence to  other  tubers,  as  carrots,  etc.— ground  potato 
may  be  used.  For  this  purpose,  the  boiled  and 
ground  potatoes  are  placed  in  flasks  (preferably  in 
the  so-called  Erlenmeyer  flasks),  and  sufficient  water 
is  added  to  form  a  thick  broth.  This  potato-broth  is 
sterilized  in  the  steam  apparatus,  and  is  then  inocu- 
lated in  the  usual  way  with  a  platinum-needle.  The 
potato-broth  can  be  made  into  a  very  good  culture- 
media  for  many  bacteria  by  the  addition  of  starch, 
sugar,  peptone,  and  beef-extract,  and  can  then  be 
very  advantageously  used  to  obtain  fractional-cul- 
tures of  certain  forms  of  bacteria. 

4.    THE  GrELATIN-CULTUKE   OF  KLEBS  AND  BEEFELD. 

Origin  from  One  Germ.  —  Moist  Chambers.  — 
When  Klebs  (I.  c.)  sought  to  fix  a  coccus  under  the 
microscope  in  order  to  directly  observe  its  division, 
and  so  follow  back  the  entire  pure  culture  to  a  sin- 
gle germ,  he  was  unable  to  do  this  in  a  fluid-drop, 
first,  because  of  the  movement  which  occurred  in 
the  drop,  and  second,  because  after  the  admission 
of  air  the  fluid  was  altered  by  the  concentration 
produced  by  evaporation.  This  also  changed  the 
value  of  the  fluid  as  a  culture-medium.  In  order 
to  prevent  the  evaporation  of  the  fluid,  or  at  least 
to  limit  it  and  to  avoid  the  motion,  instead  of  the 
ordinary  culture  -  solutions,  Klebs  used  isinglass 
as  a  culture-medium,  which  became  stiff  on  cool- 
ing. 

Now,  in  order  to  fix  a  single  coccus  present  in  the 
isinglass,  Klebs  used  the  chamber  of  von  Reckling- 
hausen  and  Geissler.  In  this  (Fig.  10)  a  tube  leads 
to  and  a  second  away  from  a  middle  room  made  of 


106 


BACTERIOLOGICAL  INVESTIGATION. 


glass  of  the  thickness  of  cover-glass,  the  upper  and 
lower  sides  of  which  almost  touch  in  the  middle,  so 


FIG.  10. 


that  here  is  formed  a  small  capillary- chamber.  If 
the  chamber  is  then  filled  with  water,  culture-fluid, 
or  liquefied  gelatin,  and  these  solutions  are  again 
poured  out,  a  capillary -drop  remains  suspended  in 
the  narrow  place.  If  these  solutions  at  the  same  time 
contain  germs,  and  in  such  numbers  that  each  drop 
holds  about  one  germ,  then,  in  many  cases,  it  will 
happen  that  the  drop  remaining  will  contain  a  germ, 
which  can  be  tolerably  well  fixed  with  a  high-power 
dry  system  and  thus  observed.  Sometimes  Klebs 
filled  the  entire  chamber  with  gelatin  or  isinglass, 
so  that  the  entrance  of  air  to  the  central  portion  was 
prevented ;  sometimes  he  left  only  one  drop  in  the 
chamber,  but  then  took  care  that  the  air  was  filtered 
through  cotton  before  its  admission. 

In  other  cases  Klebs  used,  in  place  of  the  chamber 
with  the  capillary  -  room,  a  chamber  the  walls  of 
which  ran  parallel  and  whose  side-tubes  were  placed 
somewhat  lower  than  the  upper  wall.  These  cham- 
bers (b  in  Fig.  11)  (the  description  of  which  is  after 
those  used  by  myself)  were  filled  (I.  <?.,  page  46)  by 


CULTURE-METHODS;  PURE  CULTURES. 
FIG.  11. 


allowing  the  excess  of  the  fluid  gelatin  to  run  away, 
so  that  a  thin  layer,  looking  downward,  covered  the 
upper  side  and  served  for  microscopical  examination. 
Any  germs  fixed  in  this  layer  could  be  directly  ob- 
served with  a  high  dry  lens  or  a  low  immersion  sys- 
tem, and  their  development  noted.  In  place  of  air, 
different  gases  may  also  be  admitted. 

Brefeld  *  sought  at  first  to  procure  for  fungi  an 
entirely  pure  culture-medium,  in  order  that,  proceed- 
ing from  a  single  germ,  he.  might  follow  out  without 
interruption  the  entire  development  of  the  fungus. 
These  studies  Pasteur  f  soon  undertook  upon  yeast, 
and  later,  in  1881,  Brefeld  completed  them  for  bac- 
teria, and  proved  their  usefulness  in  the  phases  of  de- 
velopment of  the  bacillus  subtilis.  "By  a  mixture 
of  the  germs  with  water  in  such  a  proportion  that  in 
a  certain  amount,  either  none  or  only  one  germ  is 
present,  the  separation  of  individual  germs,  even  of 
the  smallest  forms,  may  be  empirically  brought  about 
without  direct  observation."  "  There  results  from 
this  method  of  separation  an  equal  distribution  of 
the  individual  germs  in  the  fluid;  but  this  is  not 
always  easy  to  obtain,  and,  if  the  work  is  not  very 

*  (a)  "  Botanische  Untersuchungen  iiber  Schimmelpilze,"  Bd.  I, 
1872,  S.  10.  (&)  "  Methoden  zur  Untersuchung  der  Pilze."  "  Ver- 
handl.  der  physik.  med.  Gesellschaft  in  Wftrzbnrg,"  N.  F.,  Bd.  VIII, 
1874-'75,  S.  43.  (c)  "  Kulturmethoden  zur  Untersuchung  der  Pilze." 
"  Botanische  Untersuchungen  fiber  Schiramelpilze,"  Bd.  IV,  1881,  S.  1. 

t  "  Etude  sur  la  Mere."     "  Corapt.  rend.,"  1873,  S.  77. 


108          BACTERIOLOGICAL  INVESTIGATION. 

carefully  and  critically  undertaken,  numerous  foreign 
germs  may  be  easily  included  in  the  culture." 

Brefeld  diluted  empirically  the  fluid  containing 
the  spores  or  germs  with  water  or  fluid  culture-media 
(I.  c.)  page  49)  "  until  a  drop,  removed  with  the  point 
of  a  needle  and  transferred  to  a  slide  and  examined 
with  a  microscope,  showed  only  one  or  two  germs. 
The  slide  with  the  drop  containing  the  germ  serves 
as  the  origin  for  the  culture,  and,  on  this  account, 
has  received  the  name  of  '  slide- culture '  to  distin- 
guish it  from  other  forms  of  culture." 

If  now  a  drop  of  culture-fluid  is  added  to  this  one 
spore,  the  uninterrupted  tracing  of  the  development 
is  made  difficult  by  two  factors.  On  the  one  hand, 
the  drop  of  culture-fluid  evaporates,  and,  by  this 
change  in  its  concentration,  its  value  as  a  culture- 
medium  is  diminished ;  and,  on  the  other,  foreign 
germs  may  gain  admission.  It  is  essential,  on  this 
account,  to  prevent  the  evaporation  of  the  culture- 
drop,  and  to  inclose  it  so  as  to  prolong  the  possibility 
of  the  observation.  "  This  may  be  accomplished  in 
two  ways :  first,  by  alteration  of  the  culture-fluid, 
and,  second,  by  the  use  of  a  peculiar  slide." 

"In  order  to  prevent  evaporation  (I.  c.,  page  15), 
caraghen  or  gelatin  may  be  added  to  the  culture-fluid 
in  such  amount,  that  it  is  still  fluid  at  30°  or  35°  C., 
but  becomes  solid  when  cooled  to  15°  C.  In  these 
gelatin  solutions  the  fungi  grow  as  in  a  thin  fluid. 
Their  development  is  favored  rather  than  retarded. 
If  now  the  culture  can  be  reversed  without  danger, 
in  order  to  prevent  foreign  germs  from  falling  in  it, 
it  can  be  examined  with  a  strong  magnifying  power." 
This  can  be  done  by  placing  it  upon  a  cover-glass. 
These  reversed  cultures  are  placed  in  a  moist  jar  (Fig. 
9,  page  102)  upon  a  support  of  glass  or  zinc. 


CULTURE-METHODS;  PURE  CULTURES.        109 

If  it  is  desirable  to  avoid  the  gelatin  culture-solu- 
tions, then  a  peculiar  slide  must  be  used,  with,  which 
the  evaporation  of  the  culture-fluid  and  the  invasion 
of  foreign  germs  are  impossible,  without  the  possibil- 
ity of  a  continuous  observation  being  in  this  way  in 
the  least  prejudiced. 

For  the  observation  in  the  hanging-drop,  the  slides 
shown  in  Figs.  6  and  7  may  be  used.  A  cover-glass 
is  thoroughly  cleaned  by  a  concentrated  mineral  acid, 
alcohol,  and  ether.  Shortly  before  use  it  is  passed 
through  the  flame,  cooled,  and  protected  from  dust. 
Then  a  small,  flat  drop  of  the  sterilized  culture-solu- 
tion is  placed  in  the  center  with  a  sterilized  plati- 
num-loop. This  drop  is  inoculated  by  the  intro- 
duction on  a  platinum-needle  of  a  particle  from  a 
pure  culture.  The  cover-glass  is  reversed,  laid  over 
the  hollow  slide,  and  a  rim  of  vaseline  drawn  around 
it  to  prevent  evaporation. 

In  place  of  the  inoculation  of  the  drop  on  the  cover- 
glass,  a  sterilized  culture-solution  in  a  test-tube  may 
also  be  inoculated  with  a  pure  culture- solution  in 
such  an  amount  that,  after  thorough  mixture  by 
shaking,  a  drop  of  equal  size,  tested  as  a  dried  cover- 
glass  preparation,  shows  one  or  two  germs.  A  drop  is 
then  taken  from  this  and  prepared  on  a  cover-glass  in 
the  same  manner.  • 

Bref eld  (1.  c. ,  page  16)  rejected  this  method.  <  <  Only 
a  small  culture-drop  can  be  taken,  since  it  becomes 
globular,  and  oscillates  on  the  slightest  movement ; 
the  germ -spore  alters  its  position,  and  with  the 
stronger  magnifying  power  is  scarcely  accessible  ;  in 
short,  the  observation  is  difficult  and  incomplete. 
This  is  also  true  if  the  gelatin  culture  -  solution  is 
used." 

With  the  above-described  precautionary  measures, 


110 


BACTERIOLOGICAL  INVESTIGATION. 


FIG.  12. 


after  many  observations  with  the  hanging-drop  ar- 
ranged in  this  way,  it  may  be  used  with  great  advan- 
tage (even  for  the  smallest  forms  of  cocci,  both  in  the 
fluid  and  gelatin-drop),  to  determine  whether  a  colony 
of  bacteria,  which  with  the  low  magnifying  power 
appears  isolated,  has  developed  from  a  single  germ. 
This  has  been  described  by  Hansen,*  in  a  remarkable 
manner  for  colonies  of  yeast,  by  the  use  of  a  sus- 
pended gelatin-drop,  which  was  protected  from  evapo- 
ration. After  some  experience,  so  that  this  can  be 
used  conveniently,  the  possibility  is  in  this  way  bet- 
ter afforded  for  the  study  of  the  individual  phases  of 
the  development  of  bacteria  from  spore  to  spore  (in  a 
drop  accessible  to  the  lower  systems  of  homogeneous 
immersion),  and  for  making  parallel  experiments  with 
staining  for  spores,  which  permits  the  fixation  of  these 

individual  phases. 
Bienstock  f  even 
used  the  staining  of 
spores  directly  in 
place  of  direct  ob- 
servation, which 
seems  to  me  is  going 
quite  too  far. 

Prazmo  wski;£ 
|  '  LJ      c     iZr"  I      used  a  slide  (Fig.  12) 

to  prevent  the  ad- 
mission of  air.  This  slide,  2%  mm.  thick,  possesses 
a  circular  depression  (d  e) ;  the  circular  surface  (c) 
inclosed  by  this  ring  is  carefully  ground  smooth, 

*  "Ueber  das  Zahlen  raikroskopischer  Gegenstande  in  der  Bota- 
nik."  "  Zeitschrift  f.  wissenschaftl.  Mikroskopie,"  1884,  Bd.  I,  S.  191. 

t  "  Zeitschrift  fur  klin.  Med.,"  1884,  S.  1. 

|  "  Untersuchungen  fiber  die  Entwicklungsgeschichte  und  Fer- 
mentwirkung  einiger  Bakterien-Arten,"  1880,  S.  10. 


CULTURE-METHODS;  PURE  CULTURES.         Ill 

and  its  plane  lies  somewhat  lower  (for  bacteria  pref- 
erably only  -5  mm.)  than  the  surface  of  the  slide,  so 
that  after  laying  on  the  cover-glass  (b)  between  c  and  b 
there  is  a  place  for  a  thin  layer  of  fluid.  The  circular 
depression  extends  away  at  e  into  a  small  furrow.  A 
small  drop  of  the  sterilized  solution  is  placed  upon 
the  surface  (c)  and  is  inoculated,  or  a  drop  containing 
an  approximately  definite  number  of  bacteria  is  thus 
used.  Then  a  cover-glass  (5),  which  has  previously 
been  passed  through  the  flame,  is  applied,  is  sur- 
rounded by  vaseline  or  wax,  so  that  (to  prevent  much 
evaporation)  air  can  only  enter  by  the  furrow  e,  and 
then  finds  access  to  the  layer  of  fluid  inclosed  be- 
tween c  and  b  from  the  entire  circle.  This  layer  of 
fluid  is  accessible  in  its  entire  depth  to  the  strong 
dry  lens  or  the  weak  immersion  system.  Brefeld 
chose,  as  did  Klebs,  the  chamber  (Fig.  10)  of  von 
Recklinghausen  and  Geissler  to  fix  the  germ,  but  (I. 
c.,  c,  1881,  page  17)  the  capillary -drop  is,  in  his  opin- 
ion, too  deep  for  the  smaller  forms,  and  the  amount 
of  fluid  so  great,  that  movements  are  produced  by 
the  slightest  influences,  such  as  can  not  be  prevented 
during  the  observation,  and  which  occasion  a  shifting 
of  the  germs  out  of  reach.  The  germ  must  be  fixed 
by  reducing  the  amount  of  the  surrounding  solu- 
tion so  that  the  movements  are  at  a  minimum ;  but 
yet  the  amount  of  the  surrounding  culture-solution 
should  be  sufficient  for  the  germ  to  complete  its  full 
development.  This  is  attained  only  in  a  thin  layer 
of  fluid.  In  order  to  prepare  this  upon  the  inner 
wall  of  a  chamber,  so  that  the  highest  lenses  may 
penetrate  it,  smaller  chambers  must  be  used,  which 
have  a  small  capillary  -  room,  made  of  glass,  the 
thickness  of  the  thinnest  cover -glass,  ground  flat 
upon  both  sides  so  that  a  layer  of  equal  thickness 


112  BACTERIOLOGICAL  INVESTIGATION. 

is  formed,  in  which  it  is  possible  with  the  strong  dry 
lens  to  fix  a  germ  for  an  indefinite  length  of  time 
without  disturbance.  It  is  wise  not  to  make  the 
chamber-room  larger  than  is  necessary  to  meet  the 
technical  requirements. 

The  carefully  cleaned  chambers  are  filled  full, 
then  the  contents  allowed  to  flow  out,  and  the  single 
germs  remaining  in  the  thin  layer  of  culture-fluid  that 
adheres  on  the  inner  wall  are  selected.  The  number  of 
the  germs,  which  are  mingled  with  the  culture-fluid, 
can  be  far  greater  in  this  case.  It  is  not  necessary  to 
make  a  previous  test  to  determine  their  number,  al- 
though, if  they  are  few,  prolonged  search  is  neces- 
sary to  find  them  on  the  wall.  I  was  successful 
without  much  trouble  in  observing  for  an  unlimited 
time  .the  germs  of  bacilli  and  other  bacteria,  and  in 
following  thus  a  complete  series  of  development  as 
with  the  greater  fungi.  In  this  way  it  is  easily  pos- 
sible to  determine  the  length  of  time  which  is  neces- 
sary for  the  process  of  growth,  for  division,  and  final- 
ly for  the  entire  cycle  of  development  from  spore  to 
spore. 

The  value  of  these  chambers  for  the  examination 
of  the  fission  fungi  extends  even  to  the  smallest 
forms,  which  are  still  easily  accessible  to  the  observer 
with  the  strongest  dry  system. 

Brefeld  used  chambers  of  the  form  a  (Fig.  11), 
designed  by  Geissler.  I  used  the  form  5,  also  de- 
signed by  Geissler.  As  was  shown  by  my  experi- 
ments, it  is  possible  to  prepare  a  thin  layer  of  fluid 
in  these  chambers,  if  they  have  been  carefully  cleaned 
with  mineral  acid,  alcohol,  and  ether,  and  have  been 
sterilized.  In  place  of  the  fluid,  it  is  also  advantage- 
ous to  prepare  a  thin  layer  of  gelatin,  in  similar 
chambers  as  was  done  by  Klebs  in  1873.  In  such 


CULTURE-METHODS;  PURE  CULTURES.         113 

layers  of  gelatin  I  have  traced,  for  single  species,  the 
formation  of  colonies,  visible  to  the  naked  eye,  which 
had  developed  from  one  germ.  In  the  chamber  chosen 
by  myself,  a  one  twelfth  homogeneous  immersion-lens 
with  a  low  ocular  could  be  used. 

With  all  these  chambers  the  ordinary  warm  stage 
is,  as  a  rule,  necessary.  Up  to  this  time,  the  chambers 
with  parallel  walls  have  been  used  by  most  observers 
for  the  uninterrupted  observation  of  the  cycle  from 
spore  to  spore.  For  experimental  work,  I  prefer  the 
far  more  convenient  mode  of  observation  by  means 
of  the  hanging-drop. 

5.  METHOD  OF  DILUTION. 

Although  Brefeld  had  positively  shown  concern- 
ing mould  fungi,  and  Pasteur  concerning  yeast,  that, 
by  the  systematic  dilution  of  a  solution  containing 
micro-organisms  or  spores,  a  determined  quantity  of 
fluid  may  be  prepared  so  as  to  contain  approximately 
one  germ,  with  which  experiments  can  be  made  by 
transfers  to  sterilized  media,  Naegeli,*  in  1877,  not- 
withstanding all  the  previous  experiments,  denied 
the  possibility  of  obtaining  pure  cultures  of  bacteria. 
"  Fission  fungi  absolutely  permit  of  no  pure  culture, 
in  part  on  account  of  their  extraordinary  minuteness, 
in  part  on  account  of  their  general  diffusion  in  the 
water  and  air."  Naegeli  then  gave  a  method  of  dilu- 
tion, not  that  he  wished  to  make  hopeless  experi- 
ments to  obtain  pure  cultures  (as  was  stated  in  his 
communication  at  that  time),  but  in  order  to  prove 
the  capacity  for  multiplication  of  the  fission  fungi, 
depending  upon  the  degree  of  alteration  of  the  me- 
dium. In  this  method  he  diluted  a  fluid  containing 

*  Xaegeli  und  Schwendener,  "  Das  Mikroskop,"  2.  Aufl.,  1877,  S. 
644. 


114  BACTERIOLOGICAL  INVESTIGATION. 

a  certain  number  of  bacteria  with  a  definite  quantity 
of  sterilized  water.  Later,  Naegeli  himself  came  to 
the  conclusion  (Z.  c.,  page  646)  that  the  solution  of 
this  problem  of  equalization  could  only  be  arrived  at 
by  testing.  He  states  that  he  then  conducted  such 
empirical  experiments  in  his  studies  of  the  lower 
fungi,*  beginning  in  1871.  In  these  experiments,  he 
diluted  decomposing  urine  so  greatly  with  water  that 
each  two  drops  contained  one  germ.  By  transfers  of 
a  drop  to  a  glass  containing  a  sterilized  solution,  he 
then  succeeded  in  obtaining  a  certain  separation  of 
the  rods  and  cocci.  After  1877  he  denied  the  possi- 
bility of  obtaining  pure  cultures. 

I  find  in  Naegeli's  investigations,  unfortunately,  no 
statements  to  bring  into  harmony  these  later  commu- 
nicated results  of  1871,  with  his  complete  denial  of 
1877,  and  his  theoretical  views  concerning  the  mor- 
phology of  bacteria  and  the  origin  of  all  bacteria 
from  cocci.  Certainly  without  any  knowledge  of 
these  experiments  of  Naegeli  in  1871,  Lister  f  in  1878 
stated  that  he  had  so  diluted  sour  milk  that  a  drop 
contained  one  germ.  He  now  inoculated  sterilized 
milk  with  from  two  to  four  drops  of  the  diluted 
fluid,  and  constantly  obtained  a  souring  and  coagula- 
tion ;  less  certainly,  however,  if  he  used  only  one 
drop,  because  in  this  case  not  every  drop  in  reality 
contained  a  germ. 

Fitz:j:  also  used  in  his  experiments  on  fermenta- 
tion the  method  of  dilution  which  he  alone  desig- 
nated as  the  "  one-cell  culture." 

*  1882,  S.  13. 

t  "  On  the  Lactic  Fermentation  and  ita  Bearings  on  Pathology." 
"Transactions  of  the  Pathological  Society  of  London,"  1878,  vol. 
XXIX. 

I  "  Ueber  Spaltpilzgfthrtmgen,"  VII.  "  Berichte  der  deutschen 
chemischen  Gesellschaft,"  Bd.  XV,  1882,  S.  867. 


CULTURE-METHODS;  PURE  CULTURES.        115 

"In  order  to  obtain  a  pure  culture  of  a  fission 
fungus  which  causes  fermentation,  it  is  absolutely 
necessary  to  start  out  from  a  single  cell  as  seed.  In 
an  ordinary  impure  culture,  with  the  aid  of  a  count- 
ing-chamber, the  number  of  fission  fungi  contained  in 
one  drop  is  approximately  determined  ;  then  this 
drop  is  so  greatly  diluted  with  sterilized  distilled 
water  that,  in  from  five  to  ten  drops  of  the  diluted, 
well-mixed  fluid,  only  one  fission-fungus  cell  is  pres- 
ent ;  then  a  drop  is  added  to  each  of  a  series  of 
about  fifty  flasks,  filled  with  fluid  and  sterilized,  and 
these  are  then  placed  in  a  thermostat  at  37°  C.  Of 
the  fifty,  five  or  ten,  in  the  course  of  the  next  three 
weeks,  show  the  development  of  fungi.  In  each  one 
of  these  flasks  the  culture-fungus  is  solitary  and  pure, 
because  it  has  developed  from  a  single  cell.  Thus 
the  different  fission  fungi  which  were  contained  in 
the  unclean  culture  are  isolated." 

For  pathogenic  bacteria,  especially  for  the  anthrax 
bacilli,  Buchner*  used  the  method  of  dilution  in 
which  the  spleen-pulp  is  scraped  and  diluted  so  much 
with  sterilized  water,  that  a  single  bacterium  is  pres- 
ent in  about  10  c.  cm.  Then  the  sterilized  culture- 
solutions  are  infected  with  10  c.  cm.  of  the  fluid.  Han- 
sen  f  proceeded  in  a  similar  manner.  He  made  the 
dilution  for  yeast  so  that  2  c.  cm.  contained  one  cell. 

For  determining  also  the  number  of  germs  in  the 
original  fluid  as  well  as  in  the  diluted  solution,  a  defi- 
nite amount  is  placed  in  an  apparatus  for  numbering 
red  blood-corpuscles.  For  this  the  chamber  of  Hay- 

*  "  Ueber  die  experimentelle  Erzeugung  des  Milzbrandcontagiums 
aus  den  Heupilzen."  "  Untersuchungen  uber  niedere  Pilze  von 
Naegeli,"  1882,  S.  147. 

f  "  Ueber  das  Zahlen  mikroskopischer  Gegenstande  in  der  Bota- 
nik."  "Zeitschrift  f.  wissenschaftl.  Mikroskopie,"  Bd.  1, 1884,  S.  191. 


116 


BACTERIOLOGICAL  INVESTIGATION. 


em-BTachet  may  be  used.  This  (Fig.  7,  page  40)  con- 
sists of  a  slide  (A  B)  upon  which  rests  a  glass  plate 
(b)  provided  with  a  circular  aperture  (c). 

This  glass  plate  is  of  a  definite  thickness,  '2  mm., 
or  for  bacteria  still  better,  '1  mm.,  as  is  furnished  by 
Zeiss.  By  application  of  a  very  carefully  ground 
cover-glass  (d\  a  room  is  formed,  bounded  by  parallel 
walls,  the  height  of  which  is  exactly  *1  or  '2  mm., 
and  it  will  contain  a  definite  amount,  a  unit  of  vol- 
ume, of  the  fluid.  The  numeration  is  accomplished 
by  the  aid  of  a  crossed  ocular  micrometer.  The 
fluid  must  exactly  fill  the  room,  and  the  cover-glass 
should  fit  accurately. 

A  modification  of  this  (Fig.  13),  which  was  designed 
by  Thoma,*  permits  of  yet  finer  work.  This  is 


manufactured  by  Zeiss.    Upon  a  slide  (A  B)  an  even 
polished  glass  plate  (a)  is  fastened,  whose  circular 

*  Abbe1,  "  Ueber  Blutkdrperzahlung."  "  Sitzungsberichte  der 
Jenaischen  Gesellsch.  f.  Med.  u.  Naturwissenschaft,"  1878,  No.  29. 
Lyon  und  Thoma,  "Ueber  die  Methode  der  Blutkorperzahlung." 
Virchow's  "  Archiv,"  1881,  Bd.  LXXXIV,  S.  131. 


CULTURE-METHODS;  PURE  CULTURES.         117 

aperture  (d)  forms  the  side-walls  of  the  chamber.  In 
this  aperture  a  circular  glass  plate  (c)  is  cemented, 
which  is  so  thick  that  the  room  (e)  between  it  and  the 
superimposed  cover-glass  (b)  is  exactly  '1  mm.  high. 
This  is  the  real  counting-room  ;  a  and  c  are  carefully 
ground  parallel  to  the  surface  of  the  slide ;  in  the 
same  way  the  cover-glass  (b)  must  be  carefully  ground 
into  a  parallel  plane,  and  so  cleaned  that,  by  its  join- 
ing with  the  polished  upper  surface  of  the  chamber, 
it  forms  Newton's  color-rings,  which  remain  after  the 
removal  of  the  pressure.  The  mixture  is  made  in  a 
mixing-vessel,  which  is  to  be  also  used  for  the  first 
numbering-chamber.  Since  exact  instructions  as  to 
its  use  are  given  with  the  apparatus,  these  data  suf- 
fice. The  advantage  of  the  Thoma  chamber  is  that 
the  ocular  micrometer  is  omitted,  because  on  the  plate 
(c)  a  crossed  division  is  engraved  which  has  one  q.  mm. 
divided  into  four  hundred  quadratic  equal  fields. 

In  this  way  the  absolute  value  of  a  portion  is  de- 
termined, which  is  not  possible  when  this  is  altered 
by  each  objective  and  each  length  of  the  draw-tube, 
as  in  an  ocular  micrometer.  But,  if  the  numbering 
is  made  ever  so  exactly,  and  the  mixture  also  care- 
fully prepared,  still  an  absolute  certainty  for  the 
source  of  a  germ  is  not  reached,  either  in  the  method 
of  dilution  or  the  "  one-cell  culture."  The  error  also 
belongs  to  this  method,  as  Hansen  in  his  excellent 
communication  frankly  states,  viz.,  that  it  is  not 
certain  whether  the  number  of  cells  are  really  present 
in  the  prepared  inoculation-fluid  which  in  the  begin- 
ning are  computed  to  be  present.  It  may  also  occur 
that  not  a  single  cell  is  included,  or  that  several  are 
present,  as  was  desired. 

The  method  of  dilution  also,  in  its  best  form,  can 
only  be  conditionally  adapted  to  the  theoretical  post- 


118  BACTERIOLOGICAL  INVESTIGATION.    . 

ulate  of  the  origin  from  one  germ.  If,  further,  only 
an  approximate  guarantee  is  desired,  that  the  differ- 
ent forms  of  bacteria  contained  in  a  mixture  are  mod- 
erately separated  by  the  method  of  dilution,  large 
numbers  of  individual  experiments  must  be  made. 
The  number  of  these  was  placed  at  fifty  by  Fitz,  for 
his  individual  case ;  but  there  must  be  hundreds  and 
thousands  if  decomposing  fluids,  or  a  very  dirty  wa- 
ter or  similar  mixtures  containing  many  different 
bacteria,  are  employed. 

All  investigators,  who  have  made  use  of  the 
method  of  dilution  (on  account  of  the  limitations  of 
the  method,  but  without  the  admission  of  the  prac- 
tical points  causing  them),  have  taken  care  to  pre- 
viously prepare  the  cleanest  possible  inoculation  ma- 
terial by  the  quantity-culture,  with  the  aid  of  such 
secondary  means  as  are  dependent,  according  to  Bre- 
feld  (I.  c.,  c,  1881,  page  12),  on  the  varying  modes  of 
life  and  certain  morphological  and  physiological  pe- 
culiarities of  the  different  forms.  With  these  restric- 
tions, the  continual  dilution,  even  to  a  "  one-cell  cul- 
ture," is  very  useful  for  individual  pathogenic  organ- 
isms, still  better  for  ferment  bacteria,  the  conditions 
of  whose  existence  are  already  approximately  known, 
or  suspected  from  the  manner  of  their  spontaneous 
presence.  The  fact  first  to  be  determined  through 
pure  cultures  —  viz.,  the  biology  of  bacteria — is  in 
these  cases  provisory,  since  it  is  presupposed  that  it 
is  already  known. 

Culture-fluids  are  chosen  in  which  it  is  certain- 
ly known,  or  presumed,  that  the  respective  fermen- 
tation can  proceed.  These  are  inoculated  with  a  par- 
ticle or  drop  of  the  original  impure  material  ;  then 
the  flasks  thus  inoculated  are  placed  at  a  higher  or 
lower  temperature  ;  transfers  are  made  a  second  time 


CULTURE-METHODS;  PURE  CULTURES.         H9 

after  development  of  the  germs  has  occurred,  and  so 
on  until  an  approximately  pure  culture  of  an  organ- 
ism is  obtained,  as  in  methods  1  and  2. 

Other  flasks  are  kept  free  from  air,  if  it  is  sup- 
posed that  the  fermentation  under  consideration  pro- 
ceeds* better,  with  a  restriction  or  absence  of  the 
oxygen  of  the  air.  In  the  anaerobic  experiments 
this  will  be  more  particularly  detailed.  After  a  few 
of  these  transfers,  enumeration  and  dilution  are  made, 
and  then  the  sterilized  culture-solutions  are  inoculat- 
ed with  the  unit  of  volume  containing  the  one  germ. 

METHOD   OF  ISOLATION  BY  HEAT. 

With  these  measure-cultures  belongs  a  more  ex- 
act description  of  the  "  heating  method,"  as  it  may 
be  briefly  designated. 

In  all  cases  in  which  bacterial  development  oc- 
curs, after  a  shorter  or  longer  exposure  to  a  boiling 
temperature,  or  to  a  still  higher  temperature  (as  was 
first  shown  by  Cohn),f  it  is  always  due  to  the  pres- 
ence of  spores  forming  bacteria,  and  to  the  resist- 
ance of  the  spores  for  a  varying  length  of  time  to 
the  high  temperature.  Miquel  J  isolated  from  urine 
a  micro-organism  bacillus  urese  (that  forms  ammonia), 
by  heating  a  glass  of  water,  that  contained  its  spores, 
to  a  temperature  of  108°  C.  Bref eld  *  found  that,  for 

*Fitz,  "Ueber  Spaltpilzgahrungen,"  IX.  "Bericbte  der  deut- 
echen  chemischen  Gesellschaft,"  1884,  Bd.  XVII,  S.  1188. 

t  "  Untersuchungen  uber  Bakterien,"  Bd.  IV.  "  Die  Bakterien  und 
die  Urzeugung."  "  Beitrage  zur  Biologie  der  Pflangen,"  Bd.  II,  Heft  2, 
1876,  S.  249.  Vergl.  auch  die  Literatur  im  ersten  Abschnitt  und 
tiber  Sporenfarbung. 

I  "  Bulletin  de  la  societe  chimique  de  Paris,"  1879,  Bd.  XXXII, 
S.  127. 

*  "  Botanische  Untersuchungen  Tiber  Schimmelpilze,"  Bd.  IV, 
1881,  S.  61. 


120  BACTERIOLOGICAL  INVESTIGATION. 

the  destruction  of  the  spores  of  the  bacillus  subtilis, 
exposure  to  a  boiling  temperature  for  three  hours  was 
necessary,  or  to  a  temperature  in  the  oil-bath  of  105° 
C.  for  fifteen  minutes,  or  107°  C.  for  ten  minutes,  or 
110°  C.  for  five  minutes.  By  reducing  this  time, 
these  bacilli  can  be  obtained  in  cultures,  pure  from 
all  less  resistant  micro-organisms.  Prazmowski* 
boiled  fluids  containing  spores  for  a  longer  or  shorter 
time,  to  gain  and  retain  pure  cultures  of  bacilli  and 
clostridii,  which  form  spores.  Without  reference  to 
these  investigations,  Gunning,  f  proceeding  from  a 
mistaken  conception  of  the  method  of  culture  with 
solid  transparent  media,  has  put  forward  the  sys- 
tematic use  of  higher  temperatures  for  the  isolation 
of  bacteria.  Gunning  himself  in  this  way  also,  as  all 
before  him  had  done,  isolated  spore-forming  species. 
In  systematically  testing  this  method  by  the  use 
of  pure  cultures  of  bacteria  (both  those  forming 
spores  and  those  free  from  them),  I  discovered  what 
was  to  be  confidently  expected  from  the  biology  of 
bacteria — namely,  that,  by  systematic  heating  below, 
to,  and  above  the  boiling  point,  only  the  more  resist- 
ant forms  can  be  separated  from  the  less  resistant. 
Consequently  the  use  of  this  method  is  limited,  for 
practical  purposes,  to  the  separation  of  spore-contain- 
ing bacteria  from  those  free  from  spores.  For  this,  it 
is  to  be  recommended.  If  there  are  two  kinds  of  spore- 
forming  species  present  having  approximately  the 
same  resisting  power,  it  is  not  possible  to  obtain,  by 
a  shorter  or  longer  heating  alone,  a  sufficient  separa- 
tion. The  process  of  obtaining  by  heat  a  tolerably 

*  "  Untersuchungen  uber  die  Entwicklungsgeschichte  und  Fer- 
mentwirkung  einiger  Bakterien-Arten,"  1880,  S.  8. 

t  "  Beitrage  zur  hygienische  Untersuchung  des  Wassers."  "  Ar- 
chiv  fur  Hygiene,"  Bd.  I,  1883,  S.  335. 


CULTURE-METHODS;  PURE  CULTURES.        121 

or  quite  pure  culture  is,  as  I  have  previously  stated,* 
restricted  to  certain  cases ;  but  for  these  it  is  very  use- 
ful— e.  g. ,  it  is  the  best  method  for  obtaining  from  the 
prepared  quantity-cultures  a  separation  of  the  spore- 
containing  bacteria  from  the  spore-free  forms.  Often 
in  this  way  a  really  pure  culture  may  be  obtained. 

6.    CULTUEES  isr  CAPILLARY-TUBES,  AFTER  SALO- 

MONSEN. 

Salomonsen  f  observed  in  spontaneous  changes  in 
the  blood,  caused  by  putrefaction,  that  black  spots  of 
decomposition  appeared  (formed  by  the  reduction 
of  the  oxy haemoglobin),  which,  on  the  bottom  of  the 
vessel,  possessed  a  sharp  circular  contour,  and  higher 
up  a  more  club-shaped  form.  These  gradually  in- 
creased in  size,  became  confluent,  and  then  produced 
a  diffuse  dark  color  in  the  entire  mass  of  blood.  While 
these  spots  are  still  isolated,  according  to  Salomon- 
sen,  each  consists  of  a  single  form  of  bacteria,  which 
by  its  growth  brings  about  the  alterations  in  the  color 
of  the  blood.  Each  such  spot  is  a  colony,  a  genuine 
pure  culture  of  a  single  species. 

If  now  fresh  or  defibrinated  blood  is  drawn  into 
a  capillary-tube,  these  isolated  decomposing  spots 
also  appear  in  the  blood  in  these  tubes.  In  place 
of  the  true  capillary-tube,  lymph-tubes  or  fine  glass- 
tubes  can  be  used,  which  are  drawn  out  at  one  end  to 
a  capillary -point  (Fig.  14). 

Among  the  colonies  thus  .developed  with  a  low 

*  "  Mittheilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  Bd. 
II,  1884,  S.  330. 

t  "  Zur  Isolation  differentur  Bakterienformen."  "  Botanische 
Zeitung,"  1876,  No.  39.  "  Studier  over  Blodets  Foraadnilse,"  1877. 
"  Eine  einfache  Methode  zur  Reinkultur  verschiedener  Faulnissbak- 
terien."  "  Botanische  Zeitung,"  1880,  No.  28. 


122 


BACTEEIOLOGICAL  INVESTIGATION. 


FIG.  14. 


magnifying  power  (such  as  a  simple  lens),  small  dif- 
ferences are  observed  in  the  size, 
the  rapidity  of  development,  and  in 
the  form  (&,  c,  d,  e,  of  the  second 
cut,  Fig.  14).  Each  of  these  small 
differences  is  a  visible  indication 
that  these  different  colonies  have 
their  origin  from  different  forms  of 
bacteria. 

If  it  is  now  desired  to  transfer 
such  a  pure  culture,  then  the  tube 
is  broken  near  the  colony ;  a  previ- 
ously sterilized  platinum-needle  is 
introduced,  and  it  is  rapidly  trans- 
ferred to  a  sterilized  solution. 

The  tubes  possess  at  one  end  a 
slight  constriction  or  indentation, 
at  which  point  (a)  the  tube  is  closed 
with  cotton  or  asbestos,  while  the 
end  drawn  out  to  a  capillary-point 
is  sealed.  The  tube  thus  prepared 
is  sterilized  by  heat.  The  closed 
\a  end  is  first  broken  in  the  fluid,  then 
the  tube  is  filled  by  sucking  on  the 
end  plugged  with  cotton  (or  as- 
bestos) ;  after  its  withdrawal  it  is 
cleaned  with  alcohol,  and  then  fresh- 
ly sealed  at  the  capillary  end  or 
closed  with  varnish.  The  appearance  of  isolated  de- 
composing spots,  or,  in  general,  of  colonies  of  bac- 
teria, is  similar  in  substances,  which  spontaneously 
solidify  or  gelatinize  (as  in  blood  by  coagulation) ; 
while  in  true  solutions  such  a  sharp  isolation  of  the 
different  colonies  does  not  occur  even  in  the  capil- 
lary-tubes. 


CULTURE-METHODS;  PURE  CULTURES.        123 

In  regard  to  this  point,  then  not  completely  under- 
stood by  Salomonsen,  this  method  was  one  of  the 
most  reliable  for  producing  real  pure  cultures,  until 
a  few  years  ago. 

If  blood  is  taken  directly  from  the  vessels  of 
sound  animals  without  letting  the  air  come  in  con- 
tact with  it,  such  decomposition-spots  never  occur. 
According  to  Salomonsen,  it  is  carried  out  in  the 
following  manner  (first  part  of  Fig.  14) :  the  end  of  a 
sterilized  glass  tube,  that  has  been  drawn  out  and 
sealed,  is  introduced  into  the  vessel  which  has  been 
previously  laid  bare,  and  opened  under  antiseptic  pre- 
cautions. After  the  blood  in  the  tube  has  first  flowed 
out,  it  is  bound  fast  to  the  vessel  at  b.  Then  the 
sealed  end  is  broken  within  the  vessel  at  /,  and  the 
tube  filled  by  suction  at  the  opposite  end,  which  has 
been  plugged  with  sterilized  cotton  or  asbestos.  After 
this  the  ligatures  a,  <?,  and  d  are  tightened,  the  vessel 
is  cut  between  a  and  &,  and  between  c  and  d,  and  the 
tube  is  sealed  in  the  flame  below  b.  At  this  end,  at 
no  time  during  the  filling  of  the  tube  can  air  gain 
admission,  and  at  the  other  end  only  filtered  air 
enters.  The  tube  is  then  placed  in  a  thermostat  at 
the  temperature  of  the  blood. 

The  glass  tubes  are  first  disinfected  with  a  one 
per  mille  solution  of  sublimate,  the  sublimate  removed 
by  alcohol,  the  alcohol  by  ether,  and  the  last  evapo- 
rated by  warming.  This  method  I  have  found  to  be 
the  most  reliable  one.  This  disinfection  should  be 
performed  before  the  tubes  have  received  their  defi- 
nite form  ;  then  one  end  is  drawn  out  and  sealed  ;  the 
other  end  receives  a  slight  narrowing  or  twisting,  and 
here,  as  at  a  (second  part  of  Fig.  14),  it  is  securely 
filled  with  asbestos  or  cotton.  The  tubes,  thus  pre- 
pared, are  finally  sterilized  by  exposure  to  a  tempera- 


124  BACTERIOLOGICAL  INVESTIGATION. 

ture  of  150°  or  160°  C.  for  one  or  two  hours,  and  after 
cooling,  immediately  before  the  operation,  the  capilla- 
ry end  is  passed  once  more  quickly  through  the  flame. 
Zahn,*  in  place  of  such  glass  tubes,  uses  a  pipette 
that  will  contain  from  50  to  100  c.  cm.,  one  end  of 
which  is  drawn  out  to  a  point,  and  the  other  has  pre- 
viously received  a  constriction  at  one  point.  Then 
the  balloon  of  the  pipette  is  heated  in  the  water-bath 
or  in  a  flame.  During  the  manifold  heating  the  air 
is  displaced  by  oxygen,  carbonic  acid,  or  hydrogen, 
and  then,  during  this  process  of  heating  and  the 
consequent  expansion  of  the  gas,  both  the  ends  are 
sealed,  the  second  one  at  the  point  of  constriction. 
The  sterilization  is  accomplished  by  exposure  to  a 
high  temperature.  Since  the  air  in  the  pipette  is  ex- 
panded by  the  heating,  after  breaking  the  point  in 
the  vessel  the  filling  of  the  pipette  follows  from  the 
negative  pressure.  In  the  subsequent  sealing,  ac- 
cording to  Zahn,  fine  cracks  sometimes  occur,  on 
account  of  which  great  care  must  be  taken.  I  cover 
over  these  cracks,  immediately  after  sealing  the  ends, 
a  layer  of  sealing-wax.  If  the  blood  is  taken  from 
sound  animals,  the  clot  separates  from  the  blood-se- 
rum. Upon  the  latter  there  forms  sometimes  a  fine 
scum  of  the  smallest  fat-drops  and  altered  blood-cor- 
puscles. The  cellular  elements  gradually  disintegrate 
by  retrograde  metamorphosis.  The  granulations  dis- 
appear, but  true  bacteria  or  cocci  do  not  gain  an  en- 
trance, and  the  spots  of  decomposition  do  not  appear. 
These  blood-granulations,  like  the  bacteria,  are  im- 
mediately apparent,  and  also,  like  bacteria,  become 
cleared  up,  by  anamorphosis  of  the  protoplasm. 

*  "  Untersuchungen  tiber  das  Vorkommen  von  Fauluisskeimen 
im  Blut  gesunder  Thiere."  "  Arch.  f.  pathol.  Anatomie,"  1884,  Bd. 
OXLV,  S.  401. 


CULTURE-METHODS;  PURE  CULTURES.        125 

If  the  blood  contains  bacteria,  as  is  the  case  in 
many  infectious  diseases,  pure  cultures  of  these  may 
be  obtained  in  the  tubes  in  this  way. 

7.  THE  INFECTION-METHODS. 

In  the  consideration  of  the  strongly  obligatory 
parasites,  those  referred  to  in  the  Introduction,  as 
possessing  in  the  highest  possible  degree,  adapta- 
bility to  the  parasitic  mode  of  life,  only  the  tissues 
of  the  animal  or  vegetable  organism  would  seem 
a  priori  to  offer  the  necessary  conditions  for  their 
existence,  and  in  many  extreme  cases  only  a  certain 
form,  or  even  only  a  certain  variety  of  these  tissues, 
seems  to  be  able  to  serve  as  host  to  the  parasite. 
These  observations  lead  to  the  inoculation  of  sus- 
ceptible species  of  healthy  animals  and  plants  with 
the  parasites,  so  that  the  artificially  infected  organ- 
ism may  thus  contain  these  parasites  in  a  pure  con- 
dition. I  will  only  mention  the  known  experiments 
concerning  trichinosis,  and  the  infection  of  plants  by 
de  Bary,  van  Tieghem,  and  especially  by  Bref eld,  who 
in  this  way  more  exactly  defined  the  problems  to  be 
solved  by  the  infection-method  *— i.  e.,  "  first,  the  de- 
termination of  how  and  where  the  germs  of  the  fungus 
gained  admission  ;  then,  secondly,  the  tracing  of  the 
development  of  the  fungus,  and  the  continuous  ex- 
tension of  the  typical  disease  in  the  host  from  the 
action  of  the  germs  that  have  gained  admission.'' 

The  transmissibility  of  many  infectious  diseases, 
established  both  by  clinical  observation  and  the  facts 
of  epidemiology,  as  well  as  by  numerous  experiments 
made  after  we  had  learned  to  ascribe  many  of  these 
diseases  to  micro-organisms,  led  to  the  use  of  the 

*  "  Die  ktnstlicbe  Kultur  parasitischer  Pilze."  "  Botanische  Un- 
tersudumgen  iiber  Hefenpilze,"  Bd.  Y,  1883,  S.  1. 


126  BACTERIOLOGICAL  INVESTIGATION. 

method  of  infection  in  the  study  of  bacteria.  We 
must  here  forcibly  distinguish  two  things :  first,  the 
transfer  to  animals  of  pure  cultures  of  bacteria  de- 
rived elsewhere  for  the  determination  of  the  malig- 
nant peculiarities  of  these  germs  ;  and,  secondly,  the 
transfer  from  animal  to  animal  without  previously 
obtaining  pure  cultures  elsewhere.  For  such  infec- 
tion, almost  entirely  formerly,  and  also  largely  now, 
the  ordinary  species  of  animals  have  been  used,  which 
were  ready  at  hand  in  the  laboratory.  Koch  *  has 
shown  that  in  the  inoculation  of  field-  and  house-mice 
with  a  decomposing  fluid,  only  the  latter  died  of  a 
certain  form  of  septicaemia  caused  by  a  fine  bacillus, 
and  that,  after  several  transfers  from  mouse  to  mouse, 
the  blood  of  the  last  animal  presented  an  absolutely 
pure  culture  of  this  form  of  bacillus.  He  demon- 
strated the  same  fact  for  another  form  of  bacillus  by 
the  inoculation  of  rabbits  with  other  decomposing 
fluids.  In  these  experiments  also,  after  a  few  trans- 
fers from  animal  to  animal,  all  other  forms  of  bacteria 
originally  present  in  the  fluid  in  large  quantity  were 
eliminated,  and  the  blood  contained  a  pure  culture  of 
a  single  form  of  bacteria.  Anthrax  bacilli  kill  mice 
with  such  absolute  certainty  that,  by  the  inoculation 
of  mice  with  mixtures  of  bacteria  which  contain  the 
anthrax  bacilli,  after  a  few  transfers  these  bacteria 
can  be  obtained  pure.  Further,  Carter  and  Koch 
succeeded  in  infecting  monkeys  with  the  blood  of 
relapsing  fever,  so  that  the  blood  of  these  animals 
presented  pure  cultures  of  the  spirochsetse  of  this 
disease. 

*  "  Untersuchungen  fiber  die  Aetiologie  der  "Wundinfectionskrank- 
heiten,"  1878.  "  Zur  Untersuchung  von  pathogenen  Organismen." 
"  Mittheilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  Bd.  I,  1881, 
8.1. 


CULTURE-METHODS;  PURE  CULTURES.        127 

From  these  observations  Koch  deducted  the  im- 
portant principle  that  for  infection-experiments,  first 
of  all,  such  species  of  animals  should  be  used  as  are 
proved  to  be  susceptible  to  the  disease  under  consid- 
eration ;  and  that  animals  should  be  chosen  of  the 
same  species  as  that  in  which  the  disease  occurs.  If 
this  is  impracticable,  the  species  which  resembles 
most  closely  the  form  of  animal  in  which  the  disease 
spontaneously  appears  should  be  used.  The  techni- 
cal rules  to  be  observed  are  given  later  in  the  inocu- 
lation-experiments. 

These  infection-methods  are  to  be  used  to  obtain 
pure  cultures  in  those  cases  where  the  blood  or  the 
artificially  infected  organism  in  general  presents  a 
pure  culture  of  an  infectious  micro-organism,  which 
shows  in  the  highest  degree  parasitic  adaptability, 
and  also  in  the  study  of  all  those  infectious  diseases  in 
which  micro-organisms  have  not  yet  been  found,  but 
which  clinically  and  epidemiologically  appear  to  be 
purely  contagious.  But  in  some  of  these  diseases,  in 
many  acute  exanthemata,  such  inoculations  seem 
quite  purposeless,  because  these  diseases  are  most 
probably  confined  exclusively  to  man,  and  because 
the  supposed  micro-organisms  probably  find,  in  the 
human  body  only,  the  conditions  necessary  for  their 
existence.  In  such  cases,  naturally  the  solution  of  all 
the  questions  presented  in  the  Introduction  is  impos- 
sible, and  it  would  be  unreasonable  to  ask  here,  from 
the  bacteriological  investigation,  the  solution  of  the 
problems,  which  are  insoluble  from  the  nature  of  the 
case. 

But  in  these  cases  those  portions  of  the  problem* 
which  can  be  solved  must  be  the  more  certainly  de- 
termined, and  the  clinical  and  epidemiological  obser- 
vations the  more  critically  studied.  But  bacteriology 
9 


128  BACTERIOLOGICAL  INVESTIGATION. 

must  only  first  forego  the  solution  of  all  problems, 
when  all  possibilities  are  really  exhausted ;  for  an 
improvement  in  the  methods  sometimes  renders  pos- 
sible the  complete  solution  of  problems  seemingly 
insoluble.  As  a  most  remarkable  example  of  this, 
Koch's  inquiry  into  the  setiology  of  tuberculosis  may 
be  cited,  which  showed  that  the  experimental  pure 
culture  of  the  bacillus  tuberculosis  was  possible  out- 
side of  the  animal  organism,  in  spite  of  the  apparently 
intense  degree  of  its  parasitic  adaptation. 

8.  THE  CULTURES  UPON  TRANSPARENT  SOLID  NU- 
TRIENT MEDIA  ACCORDING  TO  KOCH. 

In  the  communication  upon  this  method  *  the 
means  were  given,  which  had  been  previously  ap- 
proved of  for  the  re- establishment  of  pure  cultures 
in  certain  cases. 

1.  The  advantages  of  the  solid  opaque  media  for 
the  isolated  culture  of  the  characteristic  pigment  bac- 
teria, after  Schroeter. 

2.  The  possibility  of  the  isolated  development  of 
pure  colonies  of  bacteria  in  blood,  and  of  the  differ- 
ential diagnosis  of  such  colonies  with  a  low  magnify- 
ing power,  after  Salomonsen. 

3.  The  advantages  of  transparent  fluid  media  for 
many  cases,  according  to  Pasteur,  Cohn,  and  Brefeld. 

4.  The  principle  of  the  development  from  one 
germ,  after  Brefeld,  Pasteur,  Lister,  Naegeli,  and 
Fitz. 

5.  In  the  use  of  this  principle  the  necessity  of  the 
local  separation,  in  order  to  give  to  each  individual 
germ  the  possibility  of  development,  isolated  and 
pure. 

*  "  Zur  Untersuchung  von  pathogenen  Organisraen."    "Mitthei- 
lungen  aus  dem  kaiserlichen  Gesundheitsamte,"  Bd.  I,  1881,  S.  1. 


CULTURE-METHODS;  PURE  CULTURES.        129 

6.  The  introduction  of  gelatin  by  Klebs  and  Bre- 
feld,  to  prevent  the  evaporation  of  the  culture-fluids. 

The  advantages  of  these  different  methods  had 
only  been  attained  separately,  even  by  Koch,  but  he 
found  the  connecting  link  which  permitted  the  union 
of  most  of  them,  and  by  this  combination  a  most 
universal  method,  and  at  the  same  time  the  most 
simple  of  all  methods,  resulted. 

Upon  solid  culture-media,  germs,  which  have  been 
intentionally  deposited  there  or  have  been  derived 
from  the  air,  develop  into  isolated  colonies.  If  this 
solid  nutrient  medium  is  opaque,  a  satisfactory  ob- 
servation is  only  possible  in  the  case  of  micro-organ- 
isms having  a  peculiarly  characteristic  growth — as, 
e.g.,  pigment-bacteria.  But  if  the  culture-medium 
is  not  only  solid,  but  likewise  transparent,  colonies 
can  be  differentiated  from  one  another  by  the  help  of 
a  low  magnifying  power,  through  peculiarities  of 
their  growth,  which  are  not  perceptible  to  the  naked 
eye,  or  a  simple  lens.  Koch  united  the  advantages 
of  the  solid  culture-media  for  the  separation  of  differ- 
ent germs  to  the  advantages  which  the  transparent 
media  possess  for  direct  microscopical  observation. 
This  simultaneous  emphasis  upon  the  solidity  and 
transparency  of  the  culture-medium  distinguishes 
Koch's  method  from  all  others. 

Koch  followed  two  quite  different  ways  in  attain- 
ing these  objects — viz.  :  he  first  chose  a  solid,  trans- 
parent culture-medium  which,  without  any  addi- 
tions, would  answer  to  these  requirements;  and, 
second,  he  produced  a  solidification  of  the  ordinary 
clear  nutrient  solutions  by  the  addition  of  gelatinizing 
substances.  The  addition  of  these  gelatinizing  sub- 
stances to  the  culture-media  led  to  the  discovery  of 
the  principle  of  solidity  and  transparency,  and  the 


130  BACTERIOLOGICAL  INVESTIGATION. 

media  thus  prepared  temporarily  preceded  the  natu- 
ral solid  transparent  media.  The  former  were  made 
by  the  addition  to  the  previously  known  normal  cult- 
ure-media, and  to  the  approved  decoctions  and 
infusions,  of  sufficient  gelatin  to  convert  these  solu- 
tions, at  the  temperature  of  the  room,  into  transpar- 
ent solid,  nutrient  substances. 

Koch  found,  if  he  inoculated  such  a  nutrient  gela- 
tin, while  it  was  still  liquid,  with  a  drop  of  fluid  con- 
taining bacteria,  that  in  the  subsequent  solidification 
the  individual  germs  were  each  surrounded  by  a 
layer  of  gelatin.  If  the  fluid  used  for  inoculation 
did  not  contain  too  many  germs,  these  remained 
sufficiently  separated  after  the  gelatin  became  solid, 
so  that  each  germ  was  isolated  at  the  point  of  its 
fixation,  and  could  there  develop  into  an  isolated 
colony.  If,  now,  the  gelatin  solution  was  allowed  to 
solidify  on  a  transparent  glass  plate,  the  development 
of  the  colonies  from  single  germs  could  be  observed 
with  the  microscope  before  they  were  perceptible  to 
the  naked  eye  or  the  simple  lens. 

Koch  did  not  use  the  gelatinizing  substances,  as 
did  Klebs  and  Brefeld,  to  prevent  evaporation.  The 
better  nutrient  capacity  was  not  a  desirable  quality, 
as  Brefeld  emphasized,  but  was  often  even  undesir- 
able, or  at  least  an  indifferent  one.  The  possibility 
of  the  reversal  of  the  nutrient  gelatin-drop  to  prevent 
the  air-infection,  also  advanced  by  Brefeld  as  desir- 
able, was  quite  unnecessary  ;  because,  upon  the  solid 
culture-media,  the  germs  originating  from  the  air 
were  also  strictly  localized  in  their  development,  so 
that  by  their  position  they  could  be  easily  distin- 
guished from  the  colonies  of  the  germs  intentionally 
inoculated.  To  this  is  added  the  further  difference 
that  both  Klebs  and  Brefeld,  in  order  to  use  the  gela- 


CULTURE-METHODS;  PURE  CULTURES.        131 

tinizing  substances  in  their  sense,  mnst  previously 
have  a  pure  culture,  which  the  first  obtained  by  the 
fractional  culture-method,  and  the  second  by  the 
method  of  dilution.  Neither  in  the  writings  of  Klebs 
nor  of  Brefeld  is  there  found  the  slightest  intima- 
tion of  an  experiment,  or  even  of  an  idea  of  using 
gelatin  to  solidify  the  solutions  for  separation  of  dif- 
ferent germs,  or  for  obtaining  pure  cultures,  as  was 
clearly  put  forward  by  Koch.* 

In  a  colony  composed  of  very  similar  organisms, 
recognized  as  growing  characteristically,  first  by  the 
microscope  and  later  by  the  naked  eye,  the  peculiari- 
ties of  each  organism  are  grouped  together.  The 
whole  habitat  of  a  colony  can  in  this  way  be  espe- 
cially valuable  for  the  differential  diagnosis  of  organ- 
isms, the  forms  of  which  are  very  similar.  The  study 
of  the  morphological  differences,  which  show  them- 
selves in  the  pure  colonies,  makes  this  bacteriological 
method  especially  useful  for  hygienic  purposes. 

The  proof  that  a  colony,  visible  with  the  naked 
eye  and  with  a  low  magnifying  power  (up  to  80  or 
120  diameters),  which  has  a  representative  and  char- 
acteristic growth,  has  developed  from  a  single  germ, 
was  not  certainly  determined  before  the  discovery 
of  this  method,  and  was  later  strikingly  brought 
out  by  Hansen  (page  110).  After  some  practice,  the 
observer  learns  to  determine  whether  a  colony  origi- 
nated from  a  single  germ,  or  whether  it  has  proceeded 
from  the  union  of  several  colonies. 

*  When  Zopf,  in  his  work  concerning  the  fission  fungi,  referred  to 
Koch's  methods  under  Brefeld's  methods  of  gelatin-culture,  he  paid 
no  regard  to  the  statements  of  either  investigator,  since  there  can 
scarcely  he  presented  greater  differences  than  exist  between  these  two 
methods. 


132  BACTERIOLOGICAL  INVESTIGATION. 


A.  TRANSPARENT  SOLID  MEDIA  MADE  BY  THE  ADDI- 
TION OF  GELATINIZING  SUBSTANCES — "  NUTRIENT 
GELATIN." 

PREPARATION  OF  NUTRIENT  GELATIN. — To  one  of 
the  tested  nutrient  solutions,  decoction,  or  infusion, 
there  is  added  pure,  finely  cut  gelatin,  preferably  for 
the  test-tube  cultures  5  per  cent  in  amount ;  but  for 
the  slide-  and  especially  the  plate-cultures,  10  per 
cent.  This  gelatin  is  allowed  to  soak  for  half  an 
hour  to  one  hour,  then  is  completely  dissolved  by 
application  of  heat.  Since  the  reaction  of  gelatin  is 
acid,  and  most  bacteria  require  either  a  neutral  or 
slightly  alkaline  reaction  for  their  growth,  the  warm 
gelatin  solution  is  neutralized,  or,  still  better,  for 
most  cases,  rendered  slightly  alkaline  with  carbonate 
of  sodium. 

The  neutralized  gelatin  solution  is  then  boiled  for 
about  one  hour  on  the  water-bath,  for  the  complete 
separation  of  the  neutralization  precipitate  and  all 
other  substances  coagulable  by  heat ;  and  after  this, 
while  still  hot,  is  filtered  through  a  moist  fluted  filter. 
The  filtrate,  after  renewed  boiling,  must,  when  cold, 
be  solid  and  clear  without  any  cloudiness.  A  tran- 
sient cloudiness  produced  in  the  cooking  by  the  phos- 
phates, and  which  disappears  after  cooling,  has  no 
significance. 

It  is  self-evident  that  no  nutrient  gelatin  offers 
to  all  bacteria  equally  favorable  conditions  for  exist- 
ence ;  but  still  it  is  desirable  to  possess  gelatinized 
solutions,  which  are  as  universally  valuable  as  possi- 
ble, so  that  they  furnish,  to  the  largest  possible  num- 
ber of  forms  of  bacteria,  conditions  sufficiently  favor- 
able, so  that  the  germs  can  develop  into  perceptible 
and  separable  colonies.  The  meat-water  peptone  gela- 


CULTURE-METHODS;  PURE  CULTURES.         133 


tin,  as  described  by  Loftier,*  supplies  this.  It  is 
prepared  in  the  following  manner  :  half  a  kilogramme 
of  good  finely  chopped  meat  is  added  to  one  litre  of 
distilled  water,  well  stirred  up  and  allowed  to  stand 
in  an  ice-chest  twenty-four  hours.  Then  this  is  fil- 
tered through  gauze,  finally  pressed  out  with  a  pe- 
culiar meat-press,  and  the  volume  of  the  fluid,  by 
the  addition  of  distilled  water,  is  restored  to  one 
litre.  To  this  meat-water  is  added  10  grammes  of 
dry  peptone,  5  grammes  of  chloride  of  sodium,  and  50 
to  100  grammes  of  the  purest  gelatin.  The  swelling 
and  solution  of  the  gelatin,  neutralization,  boiling, 
and  filtration  are  performed  as  described  above. 

I  have  obtained  the  same  object  in  a  more  simple 
manner  with  a  nutrient  gelatin,  which  consists  of 
peptone  3  per  cent,  grape-  or  cane-sugar  J  per  cent, 
and  beef  extract  J  per  cent,  with  5  to  10  per  cent  of 
gelatin.  The  addition  of  a  small 
quantity  of  sugar  to  this  otherwise 
much  less  advantageous  solution 
makes  it  available  for  the  same 
purposes.  The  boiled  and  neu- 
tralized nutrient  gelatin  must  be 
filtered  while  hot.  A  hot-water 
filter  (Fig.  15,  T)  is  most  conven- 
iently used  for  this  purpose,  in 
which  the  mantel  of  water  between 
the  glass  funnel  and  the  outer  cop- 
per wall  is  kept  warm  by  a  flame 
which  is  placed  under  the  tube  (a), 
the  lumen  of  which  communicates 
with  the  water-mantel.  It  can  be 
done  less  conveniently  by  filtering  successively  small 

*  "  Mittheilungen  au8  dem  kaiserlichen  Gesundheitsamte,"  Bd.  I, 
1881,  S.  169. 


FIG.  15. 


134:          BACTERIOLOGICAL  INVESTIGATION. 

portions  while  hot,  during  which  it  is  advantageous, 
from  time  to  time,  to  carefully  warm  the  funnel  by  a 
small  flame. 

The  neutral,  clear,  nutrient  gelatin  is  then  poured 
into  sterilized  test-tubes  to  about  one  third  their 
height  (equal  to  about  10  c.  cm.),  with  the  aid  of  a 
sterilized  funnel  or  pipette,  and  the  sterilized  cotton 
stoppers  after  this  are  again  replaced.  This  nutrient 
gelatin  in  the  test-tubes  is  sterilized  by  discontinuous 
boiling.  For  this  purpose  the  solidified  gelatin  in 
the  test-tubes  may  be  carefully  liquefied  directly  in 
the  flame,  and  then  boiled,  or  the  gelatin  may  first 
be  dissolved  by  placing  the  tubes  in  warm  water, 
and  then,  after  drying  the  glass,  it  may  be  boiled  for 
a  short  time  in  the  flame,  or  this  may  be  done  in 
the  water-bath.  This  should  be  repeated  for  about 
ten  minutes  for  four  or  five  successive  days.  If  the 
gelatin  has  stood  for  a  long  time,  so  that  it  has  begun 
to  decrease  by  evaporation,  it  must  be  once  more 
liquefied  and  boiled  before  using. 

The  nutrient  gelatin  thus  prepared  is  used  for 
slide -cultures,  plate-cultures,  and  test-tube  cultures. 

a.  Slide-Cultures. — The  gelatin  in  the  test-tubes 
is  rendered  fluid  by  heating  or  warming  in  a  water- 
bath  at  about  30°  C.,  and  the  cotton  stopper,  so  far  as 
it  projects,  is  charred  in  the  flame  before  opening, 
to  destroy  whatever  germs  of  fungi  or  bacteria  may 
have  gathered  upon  it. 

Slides  are  thoroughly  cleaned  with  mineral  acid, 
water,  alcohol,  and  ether,  and  sterilized  by  expos- 
ure for  one  or  two  hours  to  a  temperature  of  from 
150°  to  160°  C.  in  a  dry-oven.  For  this  purpose  a 
number  of  clean  slides  are  placed  in  a  small  beaker, 
over  which  a  larger  one  is  turned  to  protect  them 
from  dust  while  cooling.  For  the  reception  of  the 


CULTURE-METHODS;  PURE  CULTURES.         135 

gelatin  a  number  of  slides  are  placed,  as  nearly  hori- 
zontal as  possible,  on  a  table  or  glass  plate,  and  cov- 
ered with  a  glass  jar  to  protect  them  from  dust.  For 
this  purpose  the  apparatus  (Fig.  16)  is  most  conven- 

FIQ.  16. 


ient.  This  consists  of  a  triangle  made  of  wood,  pro- 
vided with  leveling-screws,  and  upon  the  triangle  a 
ground-glass  plate  (g)  is  laid.  This  plate,  with  the 
aid  of  a  level  (I)  and  the  screws,  is  made  horizontal. 
Under  the  glass  plate  there  is  room  to  place  a  dish 
with  ice- water,  in  order  to  thoroughly  cool  the  glass 
plate,  and  thus  the  solidification  of  the  gelatin  is 
accelerated. 

With  a  sterilized  pipette  the  fluid  gelatin  is  placed 
upon  the  slide  in  the  form  of  a  long,  thin  layer, 
which  is  a  few  millimetres  thick,  and  does  not  extend 
at  any  point  to  the  edge  of  the  slide.  If  it  is  not  pos- 
sible to  arrange  the  slide  exactly  horizontal,  then  the 
consequent  difficulty  can  be  avoided  by  dipping  the 
test-tube  in  cool  water,  or  holding  it  under  a  water- 
jet,  so  that  the  gelatin  is  so  far  cooled  that  it  is  no 
longer  a  thin  fluid,  but  has  more  consistency. 

When  these  layers  of  gelatin  have  so  far  solidi- 
fied as  to  render  the  gelatin  not  completely  solid, 
but  very  consistent,  then  they  are  inoculated  with  a 
sterilized  platinum  needle,  with  which  a  particle  of 
the  infecting  substance  or  fluid  has  been  taken  up. 
With  this,  from  three  to  five  lines  of  inoculation  are 


136  BACTERIOLOGICAL  INVESTIGATION. 

lightly  drawn  upon  the  layer  of  gelatin.  The  depth 
of  these  lines  should  not  extend  to  the  slide.  When 
the  gelatin  is  completely  solidified  the  germs  become 
fixed,  and,  if  there  are  not  too  many  present  in  one 
line,  each  is  so  far  separated  from  others  that  it  may 
develop  into  an  isolated  colony. 

The  inoculated  slides  are  placed  in  a  moist  jar 
(Fig.  9)  upon  a  bench  of  glass  or  zinc,  across  which 
are  arranged  from  two  to  four  such  slides.  Over 
these  a  second  glass  bench  is  placed  for  support  and 
protection,  and  in  this  way  several  tiers,  one  above 
the  other,  can  be  prepared  (Fig. 
17).  These  glass  benches  are  care- 
fully cleaned  and  sterilized  by 
heat  before  use.  Such  benches 
can  be  prepared  from  a  strip  of 
zinc  about  4  cm.  broad  and  14  cm.  long,  each  end 
of  which,  from  1  to  li  cm.  wide,  is  bent  over,  or 
from  a  strip  of  glass  14  cm.  long  and  4  cm.  broad, 
by  cementing  to  the  ends  small  glass  supports  with 
Canada  balsam.  Upon  these  benches,  strips  of  dry 
filter-paper  of  corresponding  size  are  laid,  and  upon 
them  the  slides  are  placed.  An  absolute  protec- 
tion against  infection  from  the  air  is  not  essential. 
The  germs  in  the  air  can  only  settle  upon  the  surface 
of  the  gelatin,  and  are  in  this  way  recognizable,  even 
if  by  chance  they  develop  into  a  colony  upon  a  line 
of  inoculation  or  in  the  immediate  vicinity.  The 
growth  of  a  colony  in  the  line  of  inoculation  is  ob- 
served with  a  dry  lens  of  low  power,  magnifying 
from  80  to  150  diameters.  If  different  forms  of 
colonies  develop  in  the  line  of  inoculation,  a  second 
inoculation  is  made  with  the  aid  of  a  low  magnifying 
power  of  15  to  20  diameters  for  the  removal  of  a 
colony.  This  transfer  is  made  with  each  colony, 


CULTURE-METHODS;  PURE  CULTURES.         137 

showing  differences  in  growth,  to  other  slides,  so 
that  after  a  few  transfers  there  is  only  a  single  form 
upon  each  slide. 

In  all  gelatin  cultures  the  following  should  be 
noted :  In  respect  to  their  behavior  toward  the  gela- 
tin, all  bacteria  can  be  practically  divided  into  those 
which  leave  the  gelatin  solid  and  those  which  liquefy 
it.  The  first  develop  in  the  interior  of  the  gelatin  in 
a  round,  oval,  or  disk  form,  etc.,  while  on  the  surface 
a  characteristic  superficial  growth  is  produced,  some- 
times in  the  form  of  a  sharply  circumscribed  circle, 
sometimes  in  leaf-  or  grape-like  arrangement,  some- 
times in  the  form  of  concentric  rings,  etc.  Transfers 
of  such  bacteria  should  be  made  both  from  the  iso- 
lated colonies  in  the  interior  and  from  the  edge  of  the 
colonies  developing  superficially. 

The  bacteria  producing  liquefaction  of  the  gelatin 
do  this  in  different  ways — sometimes  rapidly,  some- 
times only  slowly,  in  the  form  of  a  funnel.  Since, 
with  the  advance  of  the  liquefaction,  the  advantages 
of  the  solid  culture-media  are  lost,  these  forms  should 
be  separated  from  each  other  as  quickly  as  possible 
by  using  the  fewest  germs  possible  for  inoculation, 
so  that  the  liquefying  colonies  do  not  come  in  contact 
with  the  others  until  they  have  developed  sufficiently 
for  further  inoculation.  The  inoculation  should  al- 
ways be  made  from  the  edge  of  the  colony  where  the 
liquefaction  is  extending  upon  the  still  solid  gelatin, 
because  in  the  interior  of  the  liquefied  portion  a  mixt- 
ure with  other  forms  may  have  taken  place  already. 
Colonies  should  be  chosen  for  inoculation  which,  in 
the  microscopical  examination  with  a  dry  objective, 
present  a  uniform  appearance  throughout,  and  in 
which  the  characteristic  differences  are  as  clearly 
pronounced  as  possible.  In  addition  to  this,  for  con- 


138          BACTERIOLOGICAL  INVESTIGATION. 

trol,  it  is  necessary  to  make  dried  cover-glass  prepara- 
tions for  the  microscope  from  the  colonies  transferred. 
By  direct  observation  with  a  low  magnifying  dry 
objective  or  an  especial  preparation-microscope,  the 
colony  to  be  transferred  is  picked  out  with  a  sterilized 
platinum  needle.  Some  experience  is  required  for 
obtaining  the  manual  dexterity  necessary  to  remove 
only  the  exact  portions  to  be  used  for  inoculation, 
and  not  to  touch  other  portions  of  the  surface  with 
the  needle  before  or  after. 

5.  Plate-Cultures.* — The  glass  plates  necessary  for 
use  are  of  the  thickness  of  a  slide,  and  of  such  width 
that  all  points  on  their  surface  can  be  made  accessible 
for  microscopical  observation.  The  relation  of  the 
width  to  the  length  depends  upon  the  width  of  the 
stage  of  the  microscope — about  8  x  14  or  10  x  12  cm. 
These  plates  are  thoroughly  cleaned  (mineral  acid, 
water,  alcohol,  ether),  then  placed  in  a  tin  box  of  cor- 
responding size,  covered  over,  and  sterilized  for  two 
hours  at  a  temperature  of  from  150°  to  160°  C.  After 
cooling,  such  a  glass  plate  is  placed  upon  the  glass 
plate  of  the  apparatus  (Fig.  16),  and  is  protected  from 
dust  by  a  clean  glass  jar.  The  gelatin  in  a  test-tube 
is  then  liquefied  in  a  water-bath  at  30°  C.,  or  by  heat- 
ing, and  again  so  far  cooled  that  it  is  somewhat  con- 
sistent. The  firmly  fixed  cotton  stopper  is  then  loos- 
ened by  twisting  with  previously  heated  pincettes, 
so  that  its  removal  can  be  quickly  and  easily  accom- 
plished. It  is  advantageous  to  free  the  upper  portions 

*  These  were  first  demonstrated  at  the  Hygienic  Exhibition,  and 
in  au  address  by  Koch  at  the  Eleventh  German  Congress  of  Physi- 
cians at  Berlin  in  1883.  More  recently  directions  for  these  cultures 
have  appeared  from  Biedert  in  an  article  in  the  "  Deutsche  Medicinal- 
Zeitung,"  1884,  and  from  Johne,  "  Ueber  die  Koch'schen  Kein-Kul- 
turen,"  1885. 


CULTURE-METHODS;  PURE  CULTURES.         139 

of  the  cotton  plug  from  any  possibly  deposited  germs 
by  charring  it  in  the  flame.  When  the  gelatin  in  the 
test-tube  has  been  thus  prepared,  the  tube  is  held 
with  the  thumb  and  forefinger  of  the  left  hand  in  a 
direction  as  oblique  as  possible,  without  the  gelatin 
coming  in  contact  with  the  cotton.  Then  the  previ- 
ously sterilized  platinum  needle  or  loop,  while  held 
by  its  glass  rod  in  the  right  hand,  is  introduced  into 
the  material  to  be  used  for  inoculation,  thus  removing 
a  small  portion  ;  then  the  cotton  plug  is  removed  with 
the  fourth  and  fifth  fingers  of  the  right  hand,  and 
the  platinum  needle  or  loop  is  passed  into  the  lique- 
fied gelatin,  moved  to  and  fro  in  it,  wiped  off  on  the 
sides  of  the  test-tube,  removed,  and  the  cotton  plug 
is  again  quickly  replaced. 

After  this,  the  material  introduced  is  mingled  as 
equally  as  possible  with  the  gelatin  by  turning,  in- 
clining, or  gentle  shaking ;  then  the  fluid  gelatin, 
containing  the  germs  thus  separated,  is  poured  out 
upon  the  cooled  glass  plate,  and  is  spread  out  upon  it 
with  a  glass  rod  or  platinum  wire,  previously  sterilized 
by  heat  and  again  cooled.  This  plate  is  laid  upon  a 
glass  bench  in  a  moist  chamber,  and  over  it  a  second 
glass  plate  is  placed,  so  that  here  also  several  tiers  of 
plates  can  be  arranged  in  one  jar. 

While  in  the  slide-cultures  not  the  entire  mass 
of  gelatin,  but  only  the  lines  of  inoculation  and  their 
immediate  surroundings  are  used,  and  while  on  this 
account  a  relatively  large  number  of  germs  appear  for 
separation  along  a  relatively  small  line  of  inoculation, 
in  the  plate  cultures,  on  the  other  hand,  a  relatively 
small  number  of  germs  are  distributed  in  a  proportion- 
ately much  larger  quantity  of  gelatin.  Further,  the 
division  of  the  germs  in  the  still  fluid  gelatin  is  far 
better,  because  the  individual  germs  can  develop  into 


14:0          BACTERIOLOGICAL  INVESTIGATION. 

colonies,  being  fixed  in  the  solidifying  gelatin  and 
being  isolated  and  more  widely  separated  from  other 
germs.  An  enormous  number  of  germs  can  in  this 
way  be  certainly  separated  from  one  another  in  a 
single  plate-culture,  in  which  each  colony  corresponds 
to  a  single  isolated  germ,  and  in  which  at  first  but  few 
colonies  are  in  contact. 

All  colonies  which  show  differences  are  first  exam- 
ined microscopically,  and  then  from  the  same,  pure 
cultures  are  prepared  by  a  second  inoculation.  For 
this  purpose,  in  exactly  the  same  manner,  under  the 
control  of  the  microscope,  a  particle  from  a  pure  colo- 
ny is  introduced  into  new  gelatin,  and  from  this  a 
new  plate-culture  is  prepared,  in  which  then,  with  the 
exception  of  possible  air-infection,  only  this  one  or- 
ganism develops.  In  these  second  inoculations,  pure 
cultures  can  with  certainty  be  obtained.  Contamina- 
tion by  the  air-germs  can  not  be  avoided  during  the 
opening,  but  it  is  not  even  approximately  to  be  so 
much  feared  as  infection  by  imperfectly  sterilized 
instruments  and  hands,  since  these  air-germs  also  de- 
velop in  isolated  colonies.  If  infection  from  the  air 
has  taken  place  during  the  opening  and  inoculation 
of  the  test-tubes,  these  air-germs  can  naturally  de- 
velop in  the  same  manner  in  the  gelatin  as  those  in- 
tentionally introduced ;  but  their  small  number  in 
comparison  with  the  other  organisms  furnishes  a  sus- 
picion of  their  source,  and,  moreover,  not  only  a  single 
plate-culture,  but  several  should  be  made  from  the 
same  material,  so  that  one  aids  to  control  the  other. 
If  infection  from  the  air  has  taken  place  after  the 
solidification  of  the  gelatin,  this  can  be  easily  recog- 
nized by  the  position  of  the  colonies  on  the  surface. 

In  the  larger  number  of  cases  this  procedure  suf- 
fices ;  but  now  and  again  in  decomposing  fluids,  pus, 


CULTURE-METHODS;  PURE  CULTURES. 

faeces,  and  very  dirty  water,  the  number  of  germs  trans- 
ferred with  the  drop  or  particle  is  so  great  that  a  suf- 
ficient isolation  of  the  individual  germs  is  impossible, 
because  before  the  appearance  of  perceptibly  charac- 
teristic differences  in  growth  the  colonies  have  come 
in  contact  with  one  another. 

In  these  cases  the  dilution  must  be  carried  still 
further,  and  this  can  be  done  in  two  ways  :  first,  the 
material  taken  by  the  platinum  loop  can  be  brought 
into  sterilized  distilled  water,  mingled  with  it  by 
shaking,  and  from  this  greater  or  less  diluted  mixture 
a  drop  may  be  used  for  inoculating  the  fluid  gelatin. 

A  second  procedure  is  dependent  on  fractional 
cultures.  A  tube  of  sterilized  fluid  is  first  inoculated 
in  the  described  manner,  and  is  "  the  original."  After 
thorough  mixing,  a  few  small  drops — e.  g.,  five — are 
in  the  same  manner  transferred  from  this  tube  to 
another  for  "  the  first  dilution  "  (erste  Yerdunnung). 
The  original  tube  is  held  between  the  thumb  and 
index-finger  of  the  left  hand,  and  the  tube  to  be  in- 
oculated between  the  index  and  middle  finger ;  then 
the  stopper  from  the  original  is  removed  with  the 
pincettes  and  laid  to  one  side,  or  it  is  placed  between 
the  fourth  and  fifth  fingers  of  the  left  hand,  which 
then  holds  simultaneously  two  tubes  and  the  cotton 
plug.  After  this  the  plug  from  the  second  tube  is 
removed  by  the  fourth  and  fifth  fingers  of  the  right 
hand,  the  platinum  loop  is  introduced  into  the  origi- 
nal tube,  and  a  small  drop  is  transferred  into  the 
second.  By  a  to-and-fro  movement  the  drop  is  care- 
fully removed  from  the  loop,  and  then  another  drop 
is  added,  either  with  the  same  or  a  second  platinum 
loop,  previously  prepared,  and  the  same  process  is 
repeated  about  five  times.  Then  the  plug,  held  in 
the  right  hand,  is  placed  in  the  newly  inoculated 


142          BACTERIOLOGICAL  INVESTIGATION. 

tube ;  afterward  that  in  the  left  hand  is  placed  in 
the  original  tube.  Now  the  original  (equals  No.  0) 
and  the  first  dilution  (equals  No.  1)  are  ready.  Then 
a  "second  dilution"  is  made,  and  a  third,  in  exactly 
the  same  manner,  taking  the  same  number  of  drops 
from  the  culture  preceding.  Each  of  these  tubes 
furnishes  a  plate-culture  designated  by  Nos.  0,  1,  2, 
and  3,  which  are  placed  in  the  same  moist  jar  in  tiers 
(Fig.  17).  Each  of  these  cultures  controls  the  others. 

If  a  number  of  different  forms  of  bacteria  have 
been  separated,  a  plate-culture  is  made  for  each  one 
of  these,  which,  with  the  exception  of  possible  air- 
infections,  constitutes  a  pure  culture  of  one  of  the 
single  forms  present  in  the  original  mixture.  These 
inoculations  of  the  pure  cultivated  organisms  are 
repeated  often,  making  peculiarly  characteristic  colo- 
nies, as  tested  microscopically,  in  order  in  this  way 
to  eliminate  every  soluble  chemical  constituent  of  the 
first  substance. 

c.  Test- Tube  Cultures.— On  account  of  their  slight 
protection,  after  standing  a  long  time,  it  is  scarcely 
possible  to  prevent  the  infection  of  the  slide  in  plate- 
cultures  by  air-germs,  which  is  especially  incon- 
venient if  a  liquefaction  of  the  gelatin  is  brought 
about  by  them.  On  this  account  it  is  necessary  to 
renew  the  slide-  and  plate- cultures  often,  in  order  to 
avoid  the  accumulation  of  cultures  occupying  time 
and  space,  but  especially  to  preserve  certainly  pure 
a  culture  once  obtained.  In  order  to  do  this,  test- 
tube  cultures  are  employed,  in  which,  besides,  many 
peculiarities  of  growth  can  be  better  noted. 

The  test-tube,  one  third  filled  with  solidified,  ster- 
ilized nutrient  gelatin,  needs  no  other  preparation  than 
the  loosening  of  the  cotton  stopper  by  turning  with 
the  heated  pincettes  in  order  to  remove  it  quickly. 


CULTURE-METHODS;  PURE  CULTURES.         143 


FIG.  18. 


Then,  with  a  sterilized  platinum  needle,  a  particle 
from  the  pure  culture,  under  control  of  the  micro- 
scope, is  taken.  The  test-tube  is  held  in  the  left 
hand  so  that  the  mouth  looks  downward  ;  then  with 
the  fourth  and  fifth  fingers  of  the  right  hand,  or 
with  the  pincettes,  the  cotton  plug  is  removed,  and, 
with  the  platinum  needle  held  by  its  glass  rod  be- 
tween the  thumb  and  index-finger,  one  or  more 
thrusts  are  made  into  the  gelatin  (Fig.  18).  Finally, 
while  the  mouth  still  looks  downward,  the 
cotton  stopper  is  again  securely  replaced. 

The  changes  in  such  a  tube-culture  after 
the  inoculation  with  the  bacteria  vary  con- 
siderably. As  a  general  statement,  the  fol- 
lowing data  serve :  Those  forms  of  bacteria 
which  do  not  liquefy  the  gelatin  produce  a 
characteristic  surface-growth,  extending  out 
from  the  point  of  inoculation,  so  that  soon 
a  flat  or  more  prominent  head  is  formed, 
which,  in  connection  with  the  line  of  inocu- 
lation of  the  culture,  presents  the  form  of  a 
nail — "  nail-cultures  "  ("  Nagelculturen  "  of 
Friedlander)  (Fig.  19,  a). 

In  place  of  such 
a  growth,  others  develop  on 
the  surface  in  the  form  of  con- 
centric rings,  or  present  leaf- 
or  grape-formed  appearances. 
Some  show  an  intense  surface- 
growth  and  a  want  of  develop- 
ment along  the  line  of  inocu- 
lation ;  in  others  this  is  exact- 
ly reversed.  The  cultures  ap- 
pear sometimes  dry,  sometimes  mucoid 


FIG  19. 


refracting,  others  transparent. 
10 


some  are 


The  color  of  the  cult- 


144:  BACTERIOLOGICAL  INVESTIGATION. 

ures  is  very  variable.  The  gelatin  is  sometimes  col- 
ored, sometimes  not,  and  often  a  peculiar  odor  is  pro- 
duced. All  of  these  small  morphological  and  biolog- 
ical differences  are  to  be  noted,  because  they  facilitate 
the  differential  diagnosis. 

In  the  case  of  the  bacteria  that  liquefy  the  gela- 
tin, this  is  at  times  quite  gradual,  so  that  the  gela- 
tin-culture presents  a  delicate  funnel- formed  appear- 
ance (Fig.  19,  £>).  In  other  cases  it  is  more  rapid; 
in  these,  wider  funnels  are  produced  or  the  lique- 
faction extends  more  in  the  form  of  layers.  Some- 
times a  scum  or  mycoderma  forms  on  the  top  ;  in 
others  not.  Often  on  the  border-line  between  the 
liquefied  and  still  solid  gelatin  (Fig.  19,  c)  the  culture 
appears  in  the  form  of  different-shaped  clouds  ad- 
vancing forward.  Sometimes  the  liquefaction  seems 
closely  united  to  the  advances  of  the  growth  ;  many 
times  it  precedes  it,  as  if  the  liquefied  material  was 
produced  by  the  bacteria  which  extend  farther  than 
the  visible  growth  reaches.  In  the  case  of  the  bac- 
teria producing  liquefaction  also,  the  cultures  may 
present  different  colors,  and  the  gelatin  may  undergo 
change  of  color.  An  odor  may  be  produced.  These 
differences  are  collectively  to  be  noted. 

The  adaptability  of  the  ordinary  gelatinizing  sub- 
stances— as  isinglass,  caraghen,  and  gelatin — in  re- 
gard to  temperature  is  limited  to  about  25°  C.,  be- 
cause, in  the  concentration  to  be  used,  complete 
liquefaction  takes  place  at  this  temperature,  and 
then  the  advantages  of  the  solid  nutrient  media  are 
lost. 

If  it  is  desired  to  make  use  of  the  advantages  of 
the  gelatinizing  substances,  transparency  and  solid- 
ity, at  the  temperature  of  the  culture-oven,  the  vege- 
table gelatin  called  agar-agar,  obtained  from  Graci- 


CULTURE-METHODS;  PURE  CULTURES.         145 

laria  lichenoides  and  Gigartina  speciosa,  is  used  in 
place  of  gelatin. 

Instead  of  5  or  10  per  cent  of  gelatin,  1£  or  at 
most  2  per  cent  of  finely  cut  agar-agar  is  added  to 
the  solution.  This  is  allowed  to  stand  in  an  ice-chest 
for  twenty-four  hours  to  swell,  and  is  then  dissolved 
as  completely  as  possible  by  boiling.  The  swelling  can 
be  accomplished  in  a  shorter  time  by  a  slow  heat- 
ing. This  hot  solution  is  neutralized  with  carbonate 
of  soda,  and  then  cooked  two  hours  on  the  water- 
bath,  or  one  hour  in  the  steam  cylinder,  for  the  sepa- 
ration of  the  substances  precipitated  by  the  neutrali- 
zation and  those  coagulable  by  heat.  Although  the 
precipitated  material  may  have  been  previously  sepa- 
rated as  much  as  possible  by  thick  gauze,  still  these 
agar-agar  solutions  filter  very  badly,  even  in  small 
quantities  and  in  a  hot- water  filter. 

According  to  Rosenbach,  they  are  better  filtered 
through  cotton,  while  the  glass  funnel  filled  with  cot- 
ton or  glass-wool,  and  the  vessel  for  receiving  the  fil- 
trate, are  placed  in  the  steam  apparatus.  The  filling 
of  the  tubes  with  the  agar-agar  solution  and  their 
sterilization  is  done  in  the  same  way  as  with  gelatin. 
In  thick  layers  the  agar-agar  seldom  seems  so  clear 
as  does  a  layer  of  gelatin  of  equal  thickness.  The 
inoculation  of  the  plate-cultures  is  performed  as 
usual;  the  solid  agar-agar  is  liquefied  at  about  42° 
C.  in  a  water-bath,  and  the  inoculation  and  distribu- 
tion of  germs  in  the  fluid-agar  solution  is  accom- 
plished while  the  tube  is  in  part  submerged  in  warm 
water,  since  these  solutions  under  40°  C.  lose  their 
fluid  state.  At  a  temperature  above  40°  C.,  in  which 
the  liquefaction  is  more  easily  made,  the  germs  are 
injured,  so  that  the  operation  must  be  done  between 
40°  and  42°  C. 


146  BACTERIOLOGICAL  INVESTIGATION. 

The  pouring  out  of  the  inoculated  fluid  upon  the 
plate,  on  this  account,  must  be  done  as  quickly  as 
possible.  A  1£  per  cent  solution  of  agar-agar-gelatin 
is  so  solid  at  37°  C.  that  all  the  advantages  of  the 
transparent  and  solid  nutrient  media  are  still  pre- 
served at  this  high  temperature  in  the  culture-oven. 
Many  bacteria  do  not  develop  so  well  in  agar-agar 
as  in  gelatin ;  but  Rosenbach,  however,  has  clearly 
shown  the  great  superiority  'of  this  culture-medium, 
because  most  bacteria  which  liquefy  gelatin,  and 
which  therefore  produce  no  surface-growth,  do  not 
liquefy  the  agar-agar,  but,  on  the  contrary,  show  on 
it  a  very  characteristic  superficial  growth. 

The  cultures  upon  agar-agar  can  be  used  to  sup- 
plement the  cultures  in  gelatin,  and  in  this  way  the 
number  of  characteristics  which  are  presented  to  the 
naked  eye  for  differential  diagnosis  is  materially  in- 
creased. In  the  form  of  plate-cultures,  with  gelatin 
at  the  temperature  of  the  room,  with  agar-agar  for 
the  culture-oven,  Koch's  method  of  the  pure  culture 
with  the  solid  and  transparent  media  affords,  with 
the  greatest  simplicity,  the  highest  guarantee  for  the 
certain  separation  of  the  different  germs,  however 
numerous,  from  a  mixture  of  bacteria. 

IMPROVISED  MEANS. 

With  the  great  simplicity  already  arrived  at,  on 
the  purely  technical  side  of  the  investigation,  not 
much  can  be  said  in  regard  to  improvised  means, 
since  also  here  complete  certainty  must  be  preserved 
under  all  conditions.  The  experiences,  especially  in 
hygiene,  with  the  so-called  expedited  methods  have 
taught  all  unprejudiced  observers  that  only  those 
who  have  worked  out  these  methods,  and  those  who 
are  thoroughly  experienced  and  practiced  investi- 


CULTURE-METHODS;  PURE  CULTURES. 

gators,  can  lise  them  sometimes  with  advantage  and 
often  without  detriment,  while  beginners  and  inexpe- 
rienced workers,  for  whom  they  are  really  intended, 
are,  almost  without  exception,  led  by  them  into  error. 

All  who  would  really  study  bacteriological  inves- 
tigation, or  would  found  the  important  facts  on  a 
really  practical  basis,  are  by  all  means  to  be  provided 
with  a  microscope  having  an  oil-immersion  system 
and  an  Abbe  condenser,  the  most  necessary  apparatus. 
For  a  possible  series  of  experiments  on  a  more  or  less 
extensive  expedition  it  is  advisable  not  to  take  too 
much  with  you  to  perform  this  or  that  operation 
without  the  convenient  aid  of  the  laboratory  instru- 
ments. Under  these  circumstances  he  only  will  im- 
provise proper  means  who  has  had  experience  in 
laboratory  work,  and  on  this  account  can  determine 
the  limits  to  be  reached  with  safety,  or  else  these 
small  improvised  aids  must  be  used  under  definite 
guidance  as  in  other  laboratory  work  ;  but  then  they 
have  with  improvisation  nothing  more  to  do. 

In  this  sense,  in  place  of  the  gas-flame,  the  flame 
of  the  spirit-lamp  can  be  used.  The  culture-plates 
may  be  washed  with  a  1  per  mille  solution  of  subli- 
mate and  spirit,  dried  in  the  flame  of  the  spirit-lamp, 
and  sterilized  in  it  by  strongly  heating  the  surface  in 
its  entire  extent  while  the  plate  is  held  with  pincettes. 
Then  the  plate,  with  the  heated  side  up,  is  laid  upon 
a  clean  piece  of  paper,  which  lies  upon  the  table  as 
horizontal  as  possible,  and  is  covered  with  a  soup- 
plate  which  has  been  cleaned  with  sublimate  and 
spirit,  and  so  allowed  to  cool. 

If  gelatin  in  test-tubes  has  not  been  taken,  but 
a  larger  quantity  of  sterilized  gelatin  in  a  flask,  test- 
tubes  may  be  sterilized  in  the  following  manner  : 
They  are  cleaned  with  a  1  per  mille  solution  of  subli- 


148  BACTERIOLOGICAL  INVESTIGATION. 

mate  and  spirit ;  then  the  spirit  is  removed,  and  the 
tube  dried  by  holding  it  over  the  flame  of  the  spirit- 
lamp.  A  cotton  plug  is  then  shoved  a  few  centime- 
tres down  into  the  tube  with  strongly  heated  pincettes. 
Now  the  lower  portion  of  the  tube  is  heated  over  the 
flame  as  far  as  the  cotton  plug.  After  the  tube  has 
so  far  cooled  that  it  can  be  held  by  its  lower  portion, 
then  the  upper  portion  is  heated  so  thoroughly  and 
so  long  that  the  cotton  is  browned ;  when  cooled,  the 
plug  is  drawn  so  far  out  with  the  heated  pincettes  that 
it  can  be  grasped  with  the  fingers.  A  tube  thus  ster- 
ilized is  filled  one  third  full  with  gelatin,  liquefied  by 
placing  in  hot  water ;  the  plug  is  replaced,  and  then 
the  gelatin  is  again  carefully  boiled,  allowed  to  cool 
by  dipping  in  cold  water,  and  while  it  is  yet  fluid 
it  is  inoculated,  and  the  germs  thoroughly  mingled 
with  it. 

The  gelatin  is  not  poured  on  the  plate  when  it  is 
perfectly  fluid,  because  the  plate  does  not  lie  com- 
pletely horizontal ;  but  it  is  done  when  the  gelatin 
has  become  quite  consistent,  though  before  the  ap- 
pearance of  lumps. 

As  moist  chambers,  two  plates,  laid  one  above 
the  other,  may  be  used,  the  lower  one  of  which  is 
covered  with  moistened  filter-paper.  Two  pieces  of 
wood  laid  upon  this  serve  to  support  the  culture-plate 
in  place  of  the  glass  benches. 

B.     TRANSPARENT  SOLID   MEDIA,  WITHOUT  THE  ADDI- 
TION OF  GELATINIZING  SUBSTANCES — BLOOD-SERUM. 

For  some  pathogenic  bacteria  the  transparent  solid 
media,  in  the  form  of  gelatin  and  agar-agar  cultures, 
are  not  suitable.  For  these  cases  Koch  devised, 
while  he  fully  preserved  the  principle  of  the  solid- 
ity and  transparency  of  the  culture-medium,  a  quite 


CULTURE-METHODS;  PURE  CULTURES.         149 

different  way.  Koch*  had  observed  that  sterilized 
blood-serum,  when  heated  above  65°  C.,  but  before 
reaching  coagulation  temperature,  became  solid  with- 
out losing  its  transparency.  Such  solid,  but  trans- 
parent, blood-serum  was  then  introduced  as  a  nutri- 
ent medium. 

For  collection  of  the  blood,  cylindrical  vessels  pro- 
vided with  glass  stoppers,  about  29  cm.  high  and 
about  8  to  10  cm.  wide,  are  used.  These  vessels,  after 
previous  mechanical  cleaning,  are  sterilized  by  wash- 
ing with  1  per  mille  sublimate.  After  pouring  away 
the  sublimate,  the  remainder  of  it  is  removed  with 
alcohol ;  the  alcohol  is  poured  off  and  the  traces  of 
it  removed  with  ether,  and  this  last  is  evaporated  by 
warming  in  the  dry-oven.  After  removing  the  glass 
stopper,  the  blood  of  the  slaughtered  animal  is  al- 
lowed to  run  into  this  vessel.  The  neighborhood  of 
the  cut  opening  must  be  well  cleaned,  or,  at  least, 
must  be  thoroughly  moistened.  The  blood  flowing 
out  immediately  after  the  cut,  which  washes  away 
the  dirty  particles  of  the  skin,  the  fur,  and  the  cut 
hair,  is  not  preserved. 

The  vessel  is  now  filled  to  the  brim,  closed  with 
the  stopper,  and,  as  soon  as  possible,  placed  in  an 
ice-chest,  in  which  it  remains  standing  from  twenty- 
four  to  thirty  hours,  in  order  to  render  possible 
the  formation  of  a  solid  blood-clot.  If  the  vessel  is 
moved  during  the  formation  of  the  clot,  the  blood- 
corpuscles  are  mingled  with  the  serum,  which  pre- 
vents the  latter  from  becoming  completely  clear  after 
warming.  After  the  specified  rest  and  time  a  rich 
layer  of  completely  transparent,  amber-colored  serum 
forms  above  the  blood-clot.  This  is  removed  with  a 

*  "  Berliner  klinisclie  Wochenschrift,"  1882,  No.  15,  und  "Mitthei- 
lungen  aus  dem  kaiserlichen  Gesundheitsamte,"  II,  1884,  S.  48. 


150 


BACTERIOLOGICAL  INVESTIGATION. 


FIG.  20. 


sterilized  pipette  and  transferred  to  sterilized  test- 
tubes,  which  are  filled  one  third  full,  and  then  closed 
with  cotton  plugs. 

Sterilization  can  only  be  accomplished  below  the 
coagulation  temperature  of  albumen  by  discontinuous 
heating.  For  this  purpose  the  test-tubes  are  placed 
in  the  apparatus  (Fig.  2).  The  temperature  of  the 

inner  room  (b)  is  kept  at 
about  58°  C.  The  blood- 
serum  is  exposed  to  this 
temperature  on  five  or  six 
successive  days  for  about 
one  hour  daily.  This  can 
also  be  accomplished  by 
means  of  a  water-bath, 
but  less  conveniently. 
Upon  the  fluid,  sterilized  blood-serum  there  often 
forms  a  scum  of  cholesterin,  which  should  not  be  con- 
founded with  the  mycoderma  produced  by  bacteria. 

This  sterilized,  fluid  blood-serum,  which  is  often 
used  as  such,  is  placed, 
for  bringing  about  so- 
lidification, with  the 
test-tubes  inclined  at  a 
sharp  angle,  for  attain- 
ing the  greatest  possi- 
ble extent  of  surface. 
For  this  purpose  a 
double  -  walled  box, 
made  of  zinc,  provided 
with  a  glass  cover,  is 
used.  The  chamber 
between  the  double  walls  is  filled  with  water.  The 
front  side  of  this  box  can  be  lowered  by  means  of  set- 
screws  (st  in  Fig.  20,  d  in  Fig.  21).  The  box  is  then 


CULTURE-METHODS;  PURE  CULTURES.        151 

placed  at  such  an  angle  that  the  blood-serum  reaches 
to  the  upper  third  of  the  test-tube ;  but  care  should 
be  taken  that  it  does  not  come  in  contact  with  the 
cotton  plug. 

The  regulation  of  the  temperature  of  the  air- 
chamber  (&,  Fig.  21)  is  accomplished  by  a  thermome- 
ter placed  between  the  test-tubes  upon  the  floor. 
Solidification  takes  place  at  65°  C.  ;  the  higher  the 
temperature  rises  above  65°  C.,  and  the  more  it  ap- 
proaches the  coagulation  temperature  of  75°  C.,  the 
more  rapidly  solidification  occurs  ;  but  transparency 
is  more  surely  obtained  the  lower  the  temperature  is 
used.  The  serum  always  becomes  opaque  when  the 
temperature  approaches  the  point  of  coagulation.  On 
this  account  it  is  best  to  keep  the  temperature  as 
nearly  as  possible  at  65°  C.,  and  at  least  not  to  allow 
it  to  rise  above  68°  C.  The  blood  of  different  animals 
solidifies  with  different  rapidity — the  blood  of  sheep 
most  rapidly,  calves'  blood  most  slowly.  Generally, 
solidification  takes  place  in  from  one  half  to  one 
hour's  time. 

Blood-serum  that  has  been  properly  solidified  is  as 
solid  and  firm  as  hard-cooked  white  of  egg,  similar 
to  amber,  transparent,  and  only  in  the  lower  third  a 
light  milky  cloud  appears. 

During  the  heating,  water  of  condensation  formed 
on  the  upper  wall  of  the  test-tube,  which  is  coolest, 
collects  at  the  bottom  of  the  test-tube  when  it  is 
placed  upright,  and  forms,  b^  absorption  of  soluble 
substances,  a  nutritive  solution,  so  that  the  simultane- 
ous growth  upon  solid  and  fluid  nutrient  media  may 
be  observed  after  the  inoculation,  if  the  inoculation 
extends  to  the  edge  of  the  fluid. 

By  evaporation  the  serum  gradually  dries,  begin- 
ning from  above,  yet  the  middle  and  lower  portions 


152  BACTERIOLOGICAL  INVESTIGATION. 

remain  available  for  months.  Loffler  showed  that  so- 
lutions which  heighten  the  nutrient  value  of  the 
blood-serum,  when  added  in  small  amounts,  do  not 
diminish  its  capacity  for  solidifying  in  a  transparent 
form,  so  that  in  place  of  the  pure  blood  an  improved 
blood-serum  can  often  be  advantageously  used.  Lof- 
fler* adds  to  three  parts  of  blood-serum  one  part 
of  beef-infusion.  The  beef-infusion  is  prepared  ex- 
actly after  the  manner  of  Loffler's  gelatin  (page  133) ; 
1  per  cent  of  peptone,  1  per  cent  of  grape-sugar,  and 
£  per  cent  of  chloride  of  soda  is  added  ;  the  solution 
is  boiled,  neutralized  with  carbonate  of  soda,  boiled 
again  on  the  water-bath  until  the  albumen  is  com- 
pletely precipitated,  and  then  filtered.  This  bouillon 
is  sterilized  in  the  steam-cylinder,  and,  after  cooling, 
is  added  to  the  serum  ;  and  then  the  serum,  with  the 
added  bouillon,  is  sterilized  by  discontinuous  heating, 
and  solidification  is  brought  about.  It  is  self-evident 
that  other  substances  may  also  be  added — e.  g.,  solu- 
tions of  beef-extract  with  sugar,  etc.  Since  the  test- 
tube  cultures  can  not  be  directly  observed  with  the 
microscope,  blood-serum  may  also  be  allowed  to  so- 
lidify in  watch-glasses,  or  in  hollowed-out  glass  blocks 
which  are  covered  with  glass  plates  ;  but  then  the 
greater  protection  which  the  cotton  stopper  of  the 
test-tube  affords  is  lost. 

For  inoculation  of  the  test-tubes,  they  are  held  in 
the  left  hand  as  nearly  horizontal  as  possible ;  then  a 
platinum  loop  is  dipped  into  the  substance  to  be  inocu- 
lated, the  previously  loosened  cotton  plug  is  drawn 
out  with  the  fourth  and  fifth  fingers  of  the  right  hand, 
and  the  germs  are  transferred  to  the  surface  of  the 
solidified  serum  by  firmly  drawing  lines  with  the 

*  "  Mittheilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  Bd.  II, 
1884,  S.  452  und  461. 


CULTURE-METHODS;  PURE  CULTURES.         153 

platinum  loop  upon  the  same.  The  cotton  stopper  is 
then  again  replaced. 

Since  in  test-tubes,  on  the  one  hand,  a  direct  mi- 
croscopical control,  and  on  the  other  an  isolation  of 
the  germs  in  the  solidification,  is  not  possible  as  in 
gelatinizing  substances  on  slide-  and  especially  plate- 
cultures,  many  individual  experiments  (say  ten)  must 
be  made  with  each  substance,  -and,  aside  from  this, 
the  cleanest  possible  inoculating  material  must  be 
obtained.  These  two  facts  make  the  blood-serum 
cultures,  as  contrasted  with  gelatin  and  agar-agar 
cultures,  seem  incomplete.  But,  by  overcoming  this 
technical  difficulty,  it  is  possible,  on  account  of  the 
retention  of  the  principle  of  transparency  and  solidity, 
which  distinguishes  Koch's  method  so  fundamentally 
from  all  others,  to  obtain  on  solid  blood-serum  fault- 
less clean  cultures  of  obligatory  parasitic  bacteria, 
which  can  not  be  obtained  by  any  other  method. 

The  method  of  obtaining  pure  original  material 
for  the  blood-serum  cultures  needs  still  some  expla- 
nation, especially  for  the  cases  in  which,  as  in  tuber- 
culosis, the  looked-for  organism  develops  so  slowly 
that,  in  the  interval,  contingent  rivals  outnumber  it. 

In  the  preceding  microscopical  examination  of 
sections  of  different  organs  of  tissue-fluids  and  blood, 
it  is  to  be  determined  at  what  places  the  suspected 
bacteria  are  to  be  found  most  certainly,  and  in  the 
purest  condition.  After  observation  of  these  points, 
it  is  shown  that  the  removal  of  the  inoculation-ma- 
terial is  best  performed  when  it  is  taken  from  an  ani- 
mal recently  dead  or  killed. 

The  removal  is  proportionately  more  difficult  and 
uncertain  the  longer  the  time  that  has  elapsed  after 
death,  because  then  the  possibility  of  a  mixture  with 
the  rapidly  developing  micro-organisms  of  decomposi- 


154  BACTERIOLOGICAL  INVESTIGATION. 

tion  is  always  greatly  increased.  "All  preparation 
cuts,  which  do  not  come  in  contact  with  the  inoculation 
substance,  are  to  be  made,  according  to  Koch,*  with 
hot  instruments,  but  the  inoculation  mass  itself  is  re- 
moved with  cooled  scissors  and  pincettes,  or  with  the 
cooled  platinum  loop.  The  operation  should  be  al- 
ways done  with  heated  instruments,  which  should 
be  changed  every  time  when  a  new  section  is  to  be 
laid  open.  This  constant  change  of  the  instruments 
is  necessary,  that  the  contamination  which  adheres 
to  them  in  cutting  through  the  skin  and  the  super- 
ficial layers  may  not  be  Introduced  into  the  cultures." 

With  regard  to  the  previous  preparation,  it  should 
be  noted  that,  after  placing  the  animal  upon  the  dis- 
secting-table,  the  skin,  so  far  as  the  cut  is  to  extend, 
should  be  thoroughly  moistened  throughout  its  ex- 
tent with  a  1  per  mille  solution  of  sublimate,  in  order 
to  prevent  as  much  as  possible  the  scattering  of  dust, 
hair,  etc.,  in  seizing  and  cutting. 

A  number  of  knives,  scissors,  and  pincettes  are  pre- 
viously heated  in  the  flame,  and  laid  under  a  jar  to 
protect  them  against  contact  and  dust.  Then  the 
skin  along  the  line  for  the  incision — in  the  larger 
animals  with  a  hot  scalpel,  in  the  smaller  with  hot 
pincettes  and  scissors — is  cut  through  and  laid  back  on 
both  sides,  so  far  that  further  operations  will  be  un- 
impeded. With  a  second  pair  of  hot  pincettes  and 
scissors,  if  it  is  desired  to  remove  the  pleura  or  sur- 
face of  the  lung,  an  opening  is  made  in  the  wall  of 
the  thorax,  1  by  2  cm.,  and  thus  the  surface  of  the 
lung  is  laid  bare.  If  in  this  way  an  infected  portion 
is  exposed,  for  instance,  nodules  of  tubercles,  one  or 
more  of  these  nodules  is  snipped  out  with  the  cooled 
instruments.  In  order  to  release  the  bacilli  found  in 

*  "Hittheilungen,"  Bd.  II,  1884. 


CULTURE-METHODS;  PURE  CULTURES.         155 

the  tubercle  nodules,  a  nodule  is  cut  with,  a  cooled 
knife  or  scissors,  and  a  particle  from  the  interior  is 
removed  with  the  cooled  platinum  loop  and  trans- 
ferred to  the  surface  of  the  blood-serum  ;  or  a  nodule 
is  crushed  between  two  cooled  scalpels,  and  this 
crushed  mass  is  taken  with  a  platinum  needle  and 
transferred  to  the  blood-serum.  If  it  is  less  consistent, 
it  is  cut  into  with  the  cooled  knife  and  the  material 
removed  with  a  platinum  loop.  If  blood  is  to  be 
taken  from  the  heart,  after  the  skin  has  been  cut  in 
the  same  way,  the  wall  of  the  thorax  over  the  heart 
is  opened  with  hot  pincettes  and  hot  knife  or  scissors  ; 
then,  in  the  same  way,  the  pericardium  is  opened 
with  fresh  instruments.  After  this  the  apex  of  the 
heart  is  fixed  with  cooled  pincettes  and  a  cavity  opened 
with  a  scalpel,  out  of  which  the  blood  to  be  used  for 
inoculation  is  withdrawn  on  a  platinum  loop. 

If  an  organ  from  the  abdomen  is  chosen,  this  is 
removed  with  heated  instruments  after  the  abdominal 
cavity  has  been  laid  open  in  the  same  manner,  and  is 
placed  upon  a  clean  dish  or  filter-paper,  and  then 
only  the  connective- tissue  capsule  is  cut  into.  After 
this  the  edges  are  seized  with  cooled  pincettes  and  a 
deep  tear  made  in  the  organ ;  then  with  a  platinum 
needle,  out  of  the  bottom  of  the  rent,  the  tissue,  fluid, 
or  particles  are  removed. 

For  the  superficial  lymph-glands  the  skin  is  cut 
through  in  the  same  way  ;  then  the  gland  is  fixed 
with  cooled  instruments,  and  is  opened  in  situ  for 
removal  of  material  from  the  interior,  or  it  is  released 
from  its  surroundings,  laid  upon  a  glass  plate  previ- 
ously heated  and  cooled,  cut  or  torn,  and  then  fluid 
or  a  particle  of  tissue  is  taken  from  the  interior. 

If  blood  is  to  be  taken  intra  mtam  for  cultures, 
this  can  be  done  with  the  capillary-tube  of  Salomon- 


156  BACTERIOLOGICAL  INVESTIGATION. 

sen ;  also,  after  opening  a  vessel,  a  drop  of  blood  can 
be  taken  from  the  interior  with  a  platinum  loop.  It 
is  simplest  to  free  a  spot  from  hair,  clean  this  with 
brush  and  soap,  wash  it  with  a  1  per  mille  solution 
of  sublimate,  then  with  alcohol,  and  finally  remove 
the  last  particle  of  sublimate  with  ether,  after  which 
a  sterilized  needle  is  introduced  or  a  small  cut  is  made 
with  a  sterilized  knife.  The  first  few  drops  of  blood 
welling  forth  are  removed  with  a  platinum  needle, 
and  those  coming  later  are  used  for  inoculation. 

For  removal  of  a  piece  of  the  skin  in  erysipelas 
and  similar  parasitic  processes  extending  in  the  skin, 
a  spot  is  chosen  in  which  the  process  is  advancing. 
The  skin  is  cleaned  in  the  same  manner,  and  then, 
with  heated  instruments,  a  piece  is  snipped  out  which 
finally  is  still  further  reduced  in  size  or  crushed. 

If  some  time  has  already  elapsed  since  death,  the 
organs  must  be  thoroughly  cleaned  from  the  organ- 
isms of  putrefaction  adhering  to  the  exterior.  Ac- 
cording to  Koch  (I.  c.),  this  is  accomplished  by  repeat- 
edly and  thoroughly  washing  the  organ  in  a  1  per  mille 
solution  of  sublimate.  Then  with  hot  instruments, 
changed  at  each  cut,  sections  are  removed  from  the 
surface  of  the  organ,  and  only  at  a  greater  depth  the 
tissue-fluid  or  particle  is  taken. 

In  the  larger  organs — e.  g. ,  the  spleen — according  to 
Gaffky,*  after  thorough  washing  in  a  1  per  mille  sub- 
limate solution,  a  long  cut  is  made,  almost  dividing 
the  entire  organ  ;  then,  with  a  second  sterilized  knife 
upon  the  clean-cut  surface,  a  section  is  made  that  does 
not  extend  at  any  point  to  the  capsule.  Upon  this  a 
third  is  made,  and  then  out  of  the  depth  tissue-fluid 
or  a  particle  is  taken. 

*  "  Mittbeilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  1884, 
Bd.  II,  S.  386. 


CULTURE-METHODS;  PURE  CULTURES.         157 

According  to  Loffler,*  in  order  to  make  even  the 
very  unclean  organs  useful,  the  organ  is  washed  for 
ten  minutes,  with  a  continuous  motion  by  a  glass  rod, 
in  a  5  per  cent  solution  of  carbolic  acid  in  order  to 
destroy  all  the  cocci,  bacilli,  and  fungi  adhering  to 
the  surface.  For  the  destruction  of  contingent  spores 
of  bacilli,  the  organ  is  placed  for  five  minutes  in  a  1 
per  mille  sublimate  solution,  and  laid  upon  clean  fil- 
ter-paper. After  the  surface  has  become  dry,  the 
connective-tissue  capsule  is  cut  into  with  a  hot  knife, 
the  organ  is  torn  by  seizing  the  edges  of  the  cut  with 
hot  pincettes,  and  out  of  the  bottom  of  this  rent  inocu- 
lation-material is  taken. 

The  course  of  the  culture  ought  to  be  the  same  as 
in  individual  cases  which  follow  from  the  spontaneous 
presence  of  bacteria. 

In  the  case  of  the  septic  forms,  and  the  pigment 
and  ferment  bacteria,  the  object  is  always  well  at- 
tained with  plate-cultures  of  gelatin  or  agar-agar. 
Hereby  the  small  advantages  which  a  quantity -cult- 
ure possesses  are  most  favorably  employed,  es- 
pecially with  regard  to  the  heating  and  anaero- 
biosis  which  may  be  realized  in  the  plate-cultures 
also. 

Among  the  parasitic  bacteria  there  are,  with  the 
greatest  probability,  some  as  to  which  epidemiology 
furnishes  evidence  that  they  are  probably  optional 
parasites  or  optional  septic  forms,  and  can  live  en- 
tirely, or,  under  certain  conditions,  outside  of  the 
animal  organism.  These  can  be  obtained  pure  in 
gelatin  or  agar-agar  cultures. 

For  the  obligatory  parasitic  bacteria — as  are,  in  a 
greater  or  less  degree,  the  parasites  of  the  purely 
contagious  diseases — only  the  blood-serum  cultures, 
*  Ibid.<  S.  451. 


158  BACTERIOLOGICAL  INVESTIGATION. 

up  to  the  present  time,  offer  the  possibility  of  ob- 
taining pure  original  material. 

For  those  bacteria,  as  spirilla  and  spirochsetse, 
which  as  yet  it  has  been  impossible  to  cultivate 
upon  solid  nutrient  media,  or  only  badly,  and  for 
those  more  movable  parasitic  micro-organisms,  which 
possibly  may  be  the  cause  of  one  or  another  dis- 
ease, it  is  best  to  recur  to  the  tested  method  of 
dilution— the  u  one-cell  culture."  This  method  is 
exceedingly  minute  and  not  quite  so  reliable,  but 
surpasses  all  others  and  is  far  preferable,  if  other 
means  can  be  previously  used  in  order  to  secure,  if 
not  absolutely,  yet  approximately,  pure  quantity- 
cultures. 

For  other  micro-organisms,  as  mould-fungi  and 
yeast,  the  solid  nutrient  media,  both  in  the  opaque 
form  of  layers  of  potato,  of  potato-broth,  and  a  thick 
bread-broth,  are  of  value,  and  also  the  transparent 
forms  of  gelatin,  agar-agar,  and  blood- serum  cult- 
ures. Attention  must  also  be  given  to  the  reaction, 
which,  as  a  rule,  contrary  to  the  cultures  of  bacteria, 
should  be  kept  feebly  acid  ;  and  also  to  the  consider- 
ation of  the  spontaneous  presence  of  the  germs  on  the 
nutrient  media. 

While  previously  the  pure  cultures  had  only  been 
considered  a  means  intended  for  unobjectionable  posi- 
tive inoculation,  Koch  has  shown,  upon  the  ground 
of  the  biological  peculiarities  manifested  in  the  pure 
culture,  that  there  was  not  the  slightest  reason  to  be- 
lieve that  the  aetiology  of  anthrax  is  explained  by  the 
gradual  adaptation  of  a  quite  harmless  bacterium  to 
a  parasitic  mode  of  life,  but  that  the  anthrax  bacilli 
are  not  at  all  dependent  on  the  animal  body  for  the 
retention  of  their  species,  and  that  they  find,  outside 
of  the  organism,  at  least  for  a  time,  all  the  condi- 


CULTURE-METHODS;  PURE  CULTURES.        159 

tions  necessary  for  their  existence,  and  that  their 
parasitic  existence  is  only  occasional.* 

Then  Koch  succeeded  for  the  first  time  in  cultivat- 
ing, artificially  outside  of  the  animal  body,  the  para- 
site of  a  purely  infectious  disease,  tuberculosis,  f  and 
Bref eld  $  showed,  on  the  ground  of  his  investigations 
regarding  the  septic  stage  of  the  parasitic  fungus  of 
the  cereals,  that  it  was  also  possible,  by  the  use  of  the 
right  methods,  to  bring  out  from  its  parasitic  life  this 
germ — a  parasite  in  the  narrowest  sense. 

In  conclusion  to  this,  I  suggested  *  that  each  pure 
culture  is  nothing  else  than  the  septic  stage  of  a  para- 
sitic micro-organism  ;  and  likewise  de  Bary  ||  per- 
ceived the  capacity  of  parasitic  bacteria  of  being 
bred  in  dead  organic  material. 

*  "Zur  Etiologie  des  Milzbrandes."    "  Mittheilungen  aus  dem 
kaiserlichen  Gesundheitsamte,"  Bd.  I,  1881,  S.  49. 

t  "Berliner  klin.  Wochensclirift,"  1882,  No.  15. 
I  "Die  ktinstliche  Kultur  parasitischer  Pilze."    "Botanische  Un- 
tersuchimgen  uber  Hefenpilze,"  V,  1883,  S.  1. 

*  "Fortschritte  der  Medizin,"  1884,  No.  2,  S.  70. 

||  "  Vergleichende  Morphologie  und  Biologie  der  Pilze,"  1884, 
S.  526. 


11 


IV. 

INOCULATIONS  FOR  THE  DETERMINATION  OF  THE  CAUS- 
AL RELATION  OF  BACTERIA-GROWTH  TO  DECOMPO- 
SITION AND  DISEASE. 

A.  SEPTIC  BACTERIA. 

SUBSTANCES  capable  of  decomposition,  such  as  so- 
lutions of  sugar,  glycerin,  etc.,  are  prepared  accord- 
ing to  the  indications  which  the  spontaneous  presence 
of  bacteria  suggest.  The  details  of  the  preparation 
have  been  already  mentioned  in  the  description  of 
cultures  in  fluids,  as  also  the  methods  of  sterilization. 
For  the  qualitative  experiments,  test-tubes  and  small 
flasks  are  filled  with  the  solutions  ;  for  the  quantita- 
tive experiments,  flasks  containing  a  litre  and  still 
larger  are  used,  which  have  applied,  over  the  cotton 
stopper,  caps  made  of  a  double  layer  of  filter-paper. 
If  it  is  desired  to  investigate  what  degree  of  decom- 
position the  bacteria  spontaneously  bring  forth,  then 
the  solutions  receive  no  additions ;  but  if  the  sub- 
stances are  to  be  chemically  changed,  a  corresponding 
quantity  of  the  purest  carbonate  of  lime  is  added, 
in  order  to  neutralize  the  acid  formed  in  proportion 
to  their  development. 

Inoculations  are  made  by  picking  out,  with  a  pla- 
tinum needle,  under  control  of  the  microscope,  a  par- 
ticle from  a  pure  culture  in  gelatin  or  agar-agar,  and 
introducing  it  quickly  into  the  solution.  In  these 


SEPTIC  BACTERIA.  161 

cases  not  only  one  germ,  but  a  great  number  of  the 
same  germs  are  introduced,  which  in  this  way  are  im- 
mediately transferred  into  a  position  to  more  easily 
suppress  contingent  rivals  in  the  favorable  solution. 
If  the  pure  culture  was  obtained  by  the  method  of 
dilution,  as  a  one-celled  culture,  then  the  unit  of  vol- 
ume (a  drop,  a  cubic  centimetre)  with  the  hypotheti- 
cal germ  is  transferred,  by  means  of  a  sterilized  pi- 
pette, to  the  sterilized  solution. 

Since  air-infection  in  a  fluid  is  not  immediately 
recognizable,  and  the  possibility  referred  to  is  never 
entirely  excluded  from  cultures  in  fluids  and  frac- 
tional cultures,  and  since  in  some  way  such  an  acci- 
dental germ  introduced  from  the  ordinary  septic 
bacteria  may  outgrow  all  others,  a  greater  number  of 
individual  experiments  must  be  made  in  all  investi- 
gations with  fluids.  It  is  recommended  that  these 
transfers  be  made  in  a  glass  case  kept  moist. 

An  elevation  of  temperature  favors  the  growth 
and  action  of  all  septic  bacteria,  but  the  temperature 
most  favorable  to  the  different  forms  is  limited,  and 
for  many  ferment  bacteria  it  is  about  the  tempera- 
ture of  the  blood.  On  this  account,  after  the  in- 
oculation of  the  solutions,  some  must  be  kept  at 
the  temperature  of  the  room  and  others  at  a  higher 
temperature  in  the  culture-oven,  in  order  to  ap- 
proximately determine  the  favorable  temperature  in 
a  short  time. 

Control  is  followed  out  in  three  directions :  first, 
by  exposure  of  sterilized,  uninoculated  solutions  to 
the  same  external  conditions  ;  secondly,  by  the  use 
of  the  microscope ;  and,  thirdly,  by  pure  cultures. 
If  in  the  inoculated  solutions  a  change  becomes  visi- 
ble to  the  eye  by  a  cloudiness,  by  formation  of  a 
mycoderma,  or  by  the  appearance  of  air-bubbles,  the 


162  BACTERIOLOGICAL  INVESTIGATION. 

solution  is  tested  microscopically  by  a  dried  cover- 
glass  preparation. 

The  drop  of  fluid  must  be  carefully  taken  by  ster- 
ilized instruments,  a  platinum  loop  or  pipette.  Care 
should  be  taken  to  see  whether  only  one  and  the 
same  form  is  present,  and  whether  the  form  is  iden- 
tical with  that  observed  in  the  pure  culture.  Small 
differences  may  appear,  because  the  solutions  are 
more  favorable  to  the  bacteria  than  nutrient  gelatin. 
Then,  at  the  height  of  the  fermentation,  and  more 
often  toward  the  end,  sometimes  involution  -  forms 
appear,  or  there  occur,  in  a  more  typical  manner,  in 
the  individual  bacteria,  especially  in  the  bacilli,  pecul- 
iar enlargements,  which  produce  in  the  rods  whet- 
stone, spindle,  or  tadpole  forms.  If  such  appear- 
ances are  observed,  it  is  to  be  determined  whether 
these  forms  are  associated  with  the  activity  of  the 
fermentation — i.  e.,  heightened  function — or  whether, 
on  the  contrary,  they  constitute  the  first  visible  indi- 
cations of  the  total  or  partial  exhaustion  of  the  nutri- 
ent medium  ;  or  whether  they  prepare  for  the  forma- 
tion of  spores,  by  which,  in  the  exhaustion  of  the 
medium,  the  preservation  of  the  species  is  rendered 
sure. 

Concerning  this  the  microscope  alone  gives  no  in- 
formation ;  consequently,  if  these  forms  are  seen,  cult- 
ures are  made,  in  which  different  species  develop,  if 
in  the  inoculation  foreign  bacteria  have  been  includ- 
ed ;  but  only  the  inoculated  forms  develop  after  a 
really  pure  transfer. 

With  the  determination  of  the  most  advantageous 
temperature,  the  quantity  of  oxygen  most  favorable 
to  the  development  is  also  to  be  especially  observed. 
While  many  septic  bacteria — viz.,  those  producing 
oxidation  decomposition  and  the  ordinary  aerobic 


SEPTIC  BACTERIA.  163 

forms — present  no  especial  peculiarity  in  respect  to 
this,  and,  with  the  admission  of  air  both  in  fluids  and 
in  gelatinized  solutions,  can  be  easily  obtained  pure, 
other  forms  show  a  greater  intensity  of  growth  and 
action  if  the  entrance  of  air  is  not  quite  free.  When 
they  appear  spontaneously  these  bacteria  have  a  tend- 
ency to  hydrobiosis — i.  e.,  they  develop  less  on  the 
surface  and  more  in  the  interior  of  the  fluid.  In  these 
cases  the  entrance  of  oxygen  should  be  restricted ;  but 
the  oxygen  in  solution  is  always  at  the  command  of 
these  bacteria.  It  is  very  doubtful  whether  the  ex- 
treme ground  is  tenable  which  Pasteur  takes  for  the 
departure  of  his  theoretical  observations  on  decompo- 
sition— viz. , 'that  the  anaerobic  forms  are  so  constituted 
that  the  air  acts  as  a  poison,  since  in  all  these  experi- 
ments still  other  factors  come  into  play. 

Also,  the  anaerobic  bacteria  can  be  obtained  pure 
by  gelatin  and  agar-agar  cultures,  because,  in  most 
cases,  a  restriction  of  the  entrance  of  air  guarantees 
a  sufficient  intensity  of  the  development.  This  can 
be  better  attained  in  the  thick  layer  in  the  test-tube 
cultures,  and  still  better  if,  according  to  Koch,* 
upon  the  gelatin  or  agar-agar  plates  a  thin  sheet  of 
isinglass  or  mica  is  laid  which  covers  at  least  a  third 
of  the  surface  of  the  gelatin  in  the  center.  This 
should  be  applied  as  the  gelatin  begins  to  solidify. 
"  The  mica-sheet,  on  account  of  its  elasticity,  adapts 
itself  completely  to  the  surface  of  the  gelatin  and 
excludes  the  air  from  the  portion  covered."  An- 
aerobic bacteria  develop  into  colonies  under  the 
mica-plate,  while  exquisite  aerobic  forms  grow  well 
only  about  2  mm.  within  the  edge  of  the  plate,  "  so  far 

*  "  Conferenz  zur  Erorterung  der  Cholerafrage."  "Berl.  klin. 
Wochenschrift,"  1884,  No.  31,  ff.  Und  "  Deutsche  med.  Wochen- 
sohrift,"  1884,  No.  32,  ff. 


164  BACTERIOLOGICAL  INVESTIGATION. 

as  a  diffusion  of  the  air  can  take  place."  Of  the  aero- 
bic bacteria  only  extraordinarily  small  colonies,  not 
visible  to  the  naked  eye,  are  formed  under  the  mica- 
plate.  These  probably  prolong  their  existence  by 
means  of  the  oxygen  contained  in  the  gelatin,  but 
can  not  subsequently  develop  farther.  The  inocu- 
lated plate-  and  test-tube  cultures  without  these  mica- 
plates  can  be  placed  under  the  jar  of  the  air-pump, 
or  in  a  jar  in  which  the  air  has  been  replaced  as  much 
as  possible  by  carbonic  acid. 

In  this  manner  it  is  possible  to  make  available 
the  advantages  of  the  solid  and  transparent  nutrient 
media  for  obtaining  pure  cultures  also  of  the  anaero- 
bic bacteria. 

Experiments  concerning  Anaerobiosis  in  Fluids. 
— It  is  necessary  further  to  make  experiments  con- 
cerning anaerobiosis  in  fluids  in  order,  on  the  one 
hand,  to  study  the  decomposition  with  the  absence  of 
air  or  restriction  of  its  admission ;  and  on  the  other, 
also,  to  separate,  by  the  aid  of  anaerobiosis  in  measure- 
cultures,  the  anaerobic  bacteria  from  the  purely  aero- 
bic— as  was  done  by  Fitz,*  with  the  result  of  win- 
ning, by  dilution  from  this  original  material,  a  one- 
cell  culture. 

The  experiments  concerning  anaerobiosis  may  be 
divided  into  two  classes :  (a)  those  in  which  only  the 
air  is  expelled,  and  (b)  those  in  which  the  air  is  re- 
placed by  other  gases.  The  expulsion  of  the  air  can 
be  accomplished  by  boiling,  and  then  it  must  be  pos- 
sible to  make  the  inoculation  after  cooling,  so  that  in 
the  inoculation  no  air  can  gain  admission.  Either 
the  inoculation  of  the  solution  with  a  pure  culture  in 
gelatin,  or  a  one-celled  culture,  takes  place  before 

*  "Ueber  Spaltpilzgahrungen,"  IX.  "Berichte  der  deutschen 
ohemischen  Gesellschaft,"  Bd.  XVII,  1884,  S.  1188. 


SEP  TIG  BACTERIA. 


165 


FIG.  22. 


FIG.  23. 


the  expulsion  of  the  air,  and  then  the  air  must  be 
driven  out  at  a  temperature  which  can  not  be  injuri- 
ous to  the  bacteria. 

It  is  advantageous  in  the  first  case,  necessary  in 
the  second,  that  the  solution  to  be  inoculated 
be  previously  thoroughly  sterilized. 

The  simplest  order  of  experiments  for 
examination  and  for  obtaining  quantity-cult- 
ures consists  in  filling  a  flask 
(a,  Fig.  22),  having  a  long  neck, 
half  full  with  the  impure  mixt- 
ure of  bacteria  which  is  to  be 
tested  as  to  the  presence  of 
anaerobic  bacteria,  or  with  the 
sterilized,  inoculated  solution ; 
then  the  neck  is  drawn  out  nar- 
row in  the  gas-flame  at  &,  and  the  open 
end  (c)  is  joined  to  an  aspirator  or  air- 
pump.  During  the  exhaustion  of  the 
air  the  flask  (a)  is  placed  in  a  water-bath 
at  about  38°  or  40°  C.,  in  which  it  re- 
mains boiling  strongly  for  about  half 
an  hour ;  then  the  neck  at  b  is  sealed 
in  a  pointed  flame,  while  the  suction  is 
continued. 

In  this  way  a  water-hammer  quite 
free  from  air  is  obtained,  which  strikes 
strongly  upon  the  wall,  and  in  which 
the  fluid  boils  at  the  temperature  of  the 
body.  According  to  the  material,  these 
flasks  are  kept  at  the  temperature  of 
the  room,  or  in  a  culture-oven. 

Nencki  *  proceeds  in  the  following 
manner  (Fig.  23) :  The  vessel  is  filled  half  full  with 

*  "  Beitrage  zur  Biologie  der  Spaltpilze,"  1880. 


166  BACTERIOLOGICAL  INVESTIGATION. 

the  mixture  of  bacteria  or  the  inoculated  solution,  and 
afterward  closed  with  the  double-perforated  rubber 
stopper.  Through  one  opening  of  this  stopper  a  glass 
rod  (b)  passes,  which  ends  in  a  ground  stopper  that 
fits  closely  in  the  constriction  at  c,  so  that  the  con- 
tents of  the  ball  (A)  are  completely  shut  off  from  the 
fluid  above.  In  the  second  opening  a  tube  (d\  bent 
at  a  right  angle,  is  placed,  the  end  of  which  is  con- 
nected with  an  air-pump.  During  the  suction  the 
ball  (A)  is  placed  in  the  water-bath,  and  the  glass  rod 
(b)  is  withdrawn  so  that  the  stopper  rests  at  about 
a.  As  soon  as  the  air  is  removed,  which  is  indicated 
by  the  shocks  produced  by  the  boiling  and  striking 
of  the  fluid  on  the  walls  of  the  vessel,  the  glass  rod 
is  pressed  downward  by  careful  turning,  until  the  fluid 
in  the  ball  ( A)  is  completely  cut  off  by  the  stopper. 
Then,  during  the  suction,  the  tube  is  sealed  at  d  in  a 
pointed  flame,  and,  after  the  cooling  of  the  sealed 
end,  is  broken  in  a  concentrated,  recently  prepared 
alkaline  pyrogallic  solution.  After  this  solution  has 
accumulated  until  it  reaches  about  the  height  of  n 
the  tube  is  sealed  again  at  d.  The  alkaline  pyro- 
gallic solution  serves  as  an  index  for  the  surety  of 
the  closure,  as  the  air,  gaining  entrance,  would  change 
the  clear  yellow-brown  solution  into  a  dark  color. 
In  these  experiments  no  precaution  is  taken  against 
the  escape  of  the  gas.  Pasteur*  endeavored  to  obtain 
this  in  the  following  manner  (Figs.  24  and  25) :  A 
flask,  of  the  form  shown  in  Fig.  24,  is  filled  with  the 
solution  ;  the  tube  leading  into  it  is  submerged  in  a 
porcelain  vessel  filled  with  the  same  solution.  The 
fluid  in  the  flask  and  that  in  the  porcelain  dish  are 
boiled  simultaneously  for  about  half  an  hour,  in  order 

*  "  Etudes  sur  la  bi&re,"  1876,  S.  229,  ff.     "  Des  rapports  de  1'oxy- 
g£ne  avec  la  levure." 


SEPTIC  BACTERIA. 
FIG.  24. 


167 


to  expel  all  the  air  or  tlie  oxygen  of  the  air  out  of 
the  fluid.     By  the  development  of  steam  the  fluid  is 

driven  out  of  the  balloon,  but 
Fl° 25-  is  replaced  again  by  the  fluid 

in  the  porcelain  dish,  which 
has  been  rendered  free  from 
air  by  boiling.  This  repeats 
itself  many  times  during  the 
boiling  process,  and  it  is  then 
allowed  to  cool.  During  the 
cooling  I  have  found  it  advan- 
tageous to  cover  the  fluid  in 
the  porcelain  dish  with  a  layer 
of  oil,  previously  sterilized  by 
boiling  an  hour,  in  order  to 
prevent,  as  much  as  possible, 
the  subsequent  absorption  of 
oxygen.  After  complete  cool- 


168 


BACTERIOLOGICAL  INVESTIGATION. 


ing,  the  end  of  the  entrance-tube  is  submerged  in 
sterilized  quicksilver,  and  the  whole  apparatus  placed 
in  a  culture-oven.  For  control,  Pasteur  uses  flasks 
(Fig.  25)  of  double  the  size,  which  are  filled  only  half 
full,  so  that  plenty  of  oxygen  is  present.  For  the 
inoculation  of  the  flask  (Fig.  24),  the  previous  meth- 
ods can  not  be  used.  On  this  account  a  freshly  de- 
composing solution  is  placed  in  the  addition  above 
the  glass  stop-cock,  which  must  be  well  closed.  After 
the  cooling,  a  few  drops  of  the  decomposing  solution 
are  allowed  to  enter  by  quickly  opening  and  closing 
the  cock.  In  place  of  a  decomposing  solution,  I  con- 
sider it  better  to  place  in  the  small  receptacle  a  pure 
culture,  suspended  in  water  which  has  been  distilled, 
freed  from  air,  and  sterilized. 

In  order  to  allow  the  escape  of  the  changed  prod- 
ucts of  the  bacteria,  and  to  avoid  rubber  stoppers, 
Nencki  chose  the  following  apparatus  (Fig.  26, 1.  c.) : 

FIG.  26. 


The  entrance- tube  (c),  which  is  drawn  out  and  nar- 
rowed at  three  or  four  places,  is  submerged  in  the  fluid 
to  be  tested  for  the  presence  of  anaerobic  micro-organ- 
isms, or  in  the  sterilized  fluid  inoculated  with  pure 
cultures,  while  at  the  same  time  the  litre-flask  (a)  is 
freed  from  air  by  heating.  As  a  result,  the  fluid  is 


SEPTIC  BACTERIA.  169 

aspirated  into  the  apparatus  until  about  two  thirds  of 
the  flask  (a)  is  filled. 

Then  the  end  (c)  is  connected  with  an  air-pump, 
and,  while  the  flask  (a)  is  placed  in  a  water-bath,  the 
entire  apparatus  is  exhausted  of  air,  as  previously  de- 
scribed (text  for  Figs.  22  and  23).  During  the  suction 
the  tube  (c)  is  constricted  and  sealed.  The  sealed 
end  is  then  placed  in  pure  carbonic-acid  gas  or  nitro- 
gen and  broken,  and  thus  the  apparatus  is  filled  with 
this  gas  and  the  end  (c)  is  sealed  anew.  The  sealed 
end  is  then  again  broken  in  an  alkaline,  concentrated 
pyrogallic  solution,  and,  by  heating  the  apparatus,  a 
portion  of  the  gas  is  expelled  until  the  two  balls  of 
the  U-formed  tube  (b)  are  about  half  filled  with  the 
pyrogallic  solution.  Then  the  end  (c)  is  submerged 
in  quicksilver  and  the  entire  apparatus  placed  in  a 
thermostat. 

Gunning,*  doubting  the  value  of  the  previously 
described  arrangement  of  experiments,  has  claimed 
that  the  protections  used  against  the  admission  of 
air — such  as  stop-cocks,  quicksilver,  and  pyrogallic 
solutions — offer  no  absolute  certainty,  since  he  was 
able  to  determine  the  presence  of  oxygen,  as  he  be- 
lieved, by  that  reagent  which  he  thinks  most  deli- 
cate for  detecting  oxygen — viz.,  double  ferro-cyanide 
(FejTeCyJ). 

Nencki  and  Lachowicz  f  chose,  on  this  account,  the 
following  arrangement  for  experiments  (Fig.  27)  :  the 
apparatus  consists  of  two  flasks  (A  and  C\  each  con- 
taining about  250  c.  cm.,  and  one  flask  (B)  containing 
about  50  c.  cm. 

The  flask  A  serves  for  the  development  of  hydro- 

*  "Journal  f.  pract.  Chemie,"  N.  F.,  Bd.  XX,  1879,  S.  454. 
t  "Die  Anaerobiosifrage."    '*  Archiv  f.  d.  ges.  Physiologie,"  Bd. 
XXXIII,  1883. 


170 


BACTERIOLOGICAL  INVESTIGATION. 


gen  from  iron  and  sulphuric  acid,  and  for  the  forma- 
tion of  sulphate  of  iron  free  from  the  oxide.  On  this 
account,  before  the  sealing  in  of  the  funnel-tube  (a), 


Fm.  27. 


a  number  of  rolls  of  piano- wire  are  placed  in  A.  B  is 
used  for  the  reception  of  about  10  c.  cm.  of  a  solution 
of  prussiate  of  potash,  and  for  the  development  of 
the  double  ferro-cyanide  of  iron.  C  contains  the  nu- 
trient solution.  The  flasks  are  carefully  cleaned,  or, 
better,  sterilized  and  dried  by  heat,  and  then  at  d 
joined  together.  After  this,  A  is  filled  two  thirds 
full  with  distilled  water,  rendered  acid  by  a  few 
drops  of  concentrated  sulphuric  acid,  and  heated  to 
boiling.  During  the  boiling  care  should  be  taken  that 
the  steam  condensed  in  the  tube  (d}  between  A  and  B 
flows  back  into  JL,  by  placing  the  apparatus  in  an  in- 
clined position,  because  particles  of  the  oxide  of  iron 
may  be  drawn  into  the  flask  B  with  the  steam.  ' '  After 
the  water  has  boiled  for  a  few  minutes,  it  is  allowed 
to  cool  to  50°  or  60°  C.,  and  about  30  to  40  per  cent 
of  sulphuric  acid,  well  boiled,  is  poured  through  the 
funnel-tube  (a)  from  time  to  time  in  small  portions, 
by  which  an  equal  and  rapid  stream  of  hydrogen  is 
produced  that  lasts  many  hours.  Now  a  few  fresh 


SEPTIC  BACTERIA. 

crystals  of  ferro-cyanide  of  potash  are  dissolved  in 
thoroughly  boiled  water,  and  a  few  c.  cm.  are  poured 
from  this  through  the  funnel-tube  into  the  flask  B. 
While  the  oxygen  passes  through  the  ferro-cyanide 
of  potash  solution  for  about  fifteen  minutes,  the  flask 
C  is  filled  about  two  thirds  full  with  the  sterilized 
nutrient  solution,  and  then  the  flask  B  is  sealed  at  b. 
"Now  the  fluid  in  the  flask  C  is  kept  at  the  boiling- 
point  for  ten  or  fifteen  minutes  ;  after  this  the  flame 
is  removed,  and,  while  the  fluid  is  still  boiling,  the 
entrance-tube  (e)  is  submerged  in  the  quicksilver 
(E)."  The  quicksilver  is  covered  with  a  layer  of 
alkaline-pyrogallic  solution,  and  this  with  a  layer 
of  oil  or  some  other  fluid  insoluble  in  water.  The 
funnel-shaped  tube  (c)  is  now  closed  with  a  wax  stop- 
per, and  then  the  hydrogen  gas  escapes  through  the 
quicksilver  closure  at  J57,  and  thus  expels  all  the  air 
from  this  portion  of  the  apparatus.  The  wax  stopper 
is  removed  when  the  nutrient  solution  in  C  is  cooled 
to  about  30°  C.,  and  it  is  then  inoculated  with  a  few 
drops  of  the  mixture  of  bacteria  to  be  tested,  or  of 
the  pure  culture  suspended  in  sterilized  water.  The 
neck  at  c  is  dried  with  filter-paper  and  sealed.  In 
order  to  fill  the  tube  (a)  with  hydrogen,  some  iron 
wire  is  likewise  placed  in  it,  and  this  tube  (at  a)  is 
also  sealed.  Now,  in  the  entire  apparatus  the  air 
has  been  displaced  by  hydrogen.  "In  this  manner, 
throughout  the  entire  extent  of  the  apparatus,  is  a 
stream  of  hydrogen  passed,  and  the  development  of 
the  gas  continues  for  some  hours  after  all  the  en- 
trance-tubes are  sealed.  By  inclination  of  the  appa- 
ratus some  of  the  solution  of  oxide  of  iron  is  poured 
out  from  A  into  B ;  the  resulting  precipitate  of  the 
double  ferro-cyanide  of  iron  is  completely  white,  and 
does  not  alter  its  color  even  after  long  standing." 


172  BACTERIOLOGICAL  INVESTIGATION. 

Since  the  question  of  anaerobiosis  is  by  no  means 
settled  in  all  respects,  the  direction  of  the  investiga- 
tion may  be  guided  by  the  fact  that  while  Pasteur, 
Nencki,  and  most  investigators  accept  anaerobiosis  as 
proved,  Gunning  denies  the  same,  and  designates  it 
as  a  phantasy.  In  the  experiments,  for  this  reason, 
it  is  especially  necessary  that  a  simple  hydrobiosis 
should  not  be  accepted  as  anaerobiosis,  but  that  spe- 
cial experiments  be  undertaken  for  this. 

If  anaerobiosis  is  recognized,  it  is  to  be  determined 
whether  this  is  the  cause  of  the  decomposition  (Pas- 
teur), or  whether  it  has  a  more  insignificant  relation 
to  the  course  of  the  decomposition.  As  for  myself,* 
the  entire  question  seems  to  me  to  depend  upon  the 
recognition  of  the  possibility  of  anaerobiosis. 

B.  PARASITIC  BACTERIA. 

By  the  inoculation  of  susceptible  animals  with 
pure  cultures  (vide  Infection-Methods,  page  125)  it  is 
directly  shown  that  micro-organisms  present  in  a  dis- 
eased process  are  really  the  conditio  sine  qua  non  of 
the  respective  infectious  disease.  The  indirect  meth- 
ods for  the  investigation  of  the  aetiology,  also  the 
experimental  investigations  acting  locally,  all  tend 
only  to  this  point.  A  restriction  is  often  placed  here 
to  the  methods.  In  studying  the  statistics,  also  large 
in  numbers,  we  must  here  halt,  and  consider  the  fact 
that  in  the  valuation  of  such  great  numbers  the  value 
of  the  conclusion  is  very  dependent  upon  how  the 
statistics  have  been  collected,  since  the  same  mate- 
rial has  been  brought  forward  repeatedly  with  the 

*  **  Mittheilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  Bd. 
II,  1884,  S.  345;  und  "Ueber  die  Zersetzungen  der  Milch  irod  die 
biologischen  Grundlagen  der  Gahrungsphysiologie."  "  Deutsche  med. 
Wochenschrift,"  1883,  Nos.  48  bis  50. 


PARASITIC  BACTERIA.  173 

same  resoluteness,  sometimes  to  support  the  soil-water 
theory  of  disease,  sometimes  the  drinking-water  the- 
ory, sometimes  for  the  explanation  of  disease  as  of 
a  contagious  and  sometimes  as  of  a  miasmatic  nature. 

It  would  be  a  sad  error  to  underestimate  the  sig- 
nificance of  the  setiological  investigation  directed  to 
the  study  of  the  accessory  causes  of  disease.  This 
investigation  often  furnishes  valuable  points  for  prac- 
tical treatment  by  the  denial  of  other  facts,  but  it  is 
also  wrong  to  ascribe  to  the  results  of  this  investiga- 
tion the  only  value,  and  to  regard  the  experiments 
on  animals  as  showing  nothing  concerning  the  natu- 
ral mode  of  infection.  It  would  be  well  if  one  now 
and  then  recalled  the  often  serious  defects  of  the 
methods  with  which  the  indirect  investigation  into 
aetiology  must  be  conducted,  and  in  which  the  cer- 
tainty of  the  conclusions  drawn  are  often  sharply 
contrasted  with  the  uncertainty  of  the  material  from 
which  the  observations  are  made. 

But  we  must  also  avoid  prejudice  in  the  experi- 
ments on  animals.  The  experimenter  who,  possess- 
ing susceptible  animals,  completely  masters  the  ex- 
periments, easily  falls  into  the  error  of  believing  too 
strongly  in  contagion,  and  of  concluding  too  much 
from  his  direct  results  as  to  the  natural  method  of 
infection. 

The  problem  in  the  experiments  on  animals  is  in 
reality  a  twofold  one,  but  in  the  division  of  which, 
nevertheless,  a  clear  dividing-line  can  scarcely  be 
drawn.  In  the  first  place,  it  is  to  be  determined  by 
direct  inoculation  experiments  whether  a  form  of 
bacteria  is  pathogenic  or  not,  and  whether  it  is  the 
cause  of  a  disease  or  a  possible  accompaniment.  In 
these  experiments  it  is  at  the  same  time  to  be  de- 
termined whether  the  bacteria,  after  the  artificial  in- 


174          BACTERIOLOGICAL  INVESTIGATION. 

fection,  are  distributed  and  arranged  in  the  same 
way  as  when  the  disease  appears  spontaneously  in 
the  body,  so  as  to  explain  clearly  all  clinical  symp- 
toms and  pathological  lesions.  Secondly,  we  should 
endeavor  as  nearly  as  possible  to  determine  the  method 
of  infection  which,  from  the  clinical,  anatomico-patho- 
logical, and  general  epidemiological  observations,  is 
shown  to  be  the  method  of  the  natural  infection  in 
the  spontaneous  appearance  of  an  epidemic  disease. 
The  following  methods  for  infection  can  be  used : 

Inhalation  Experiments. — The  inhalation  experi- 
ments are  performed  by  placing  the  animals  in  a  spa- 
cious box,  the  air  of  which  is  mingled  with  organisms. 
This  is  done  by  means  of  a  steam-dispersing  appara- 
tus, or,  less  conveniently,  by  a  hand-spray.  Either 
the  apparatus  is  allowed  to  act  for  a  short  time — 
twenty  minutes  to  an  hour — and  in  this  case  pure  cult- 
ures, suspended  in  large  numbers  in  sterilized  water, 
are  used,  or  the  dispersion  is  continued  for  a  longer 
time,  and  then  the  bacteria  are  mingled  in  a  corre- 
spondingly larger  mass  of  water.  Since,  in  natural 
infection  by  the  breath,  spores  probably  play  an  im- 
portant role,  this  is  to  be  taken  into  consideration 
in  the  experiments,  and  many  of  the  differences  con- 
cerning the  negative  or  positive  results  in  the  pro- 
duction of  tuberculosis  by  inhalation  of  tubercular 
sputum  can  be  traced  back  to  this.  According  to 
Veraguth,*  10  grammes  of  purulent  tubercular  spu- 
tum are  rubbed  up  with  500  grammes  of  water.  This 
mixture  is  filtered — to  remove  the  accompanying  ma- 
terial held  in  suspension,  which  might  produce  an 
inflammation  of  the  lung- tissue  by  its  mechanical  or 
chemical  action — through  two  thick  layers  of  flannel, 

*  "  Experimentelle  Untersuchungen  tlber  Inhalations-Tubercu- 
lose."  "Arch.  f.  experim.  Pathologie,"  1883,  Bd.  17. 


PARASITIC  BACTERIA.  175 

or,  according  to  Weichselbaum,*  through  absorbent 
cotton. 

In  inhalations  through  a  tracheal  fistula,  precau- 
tions should  be  taken  to  avoid  the  possible  simulta- 
neous infection  from  the  wound.  If  it  is  desired  to 
see  what  action  bacteria  produce  when  they  are  in- 
troduced into  the  trachea  in  any  way,  according  to 
Kussner,f  the  trachea  may  be  laid  bare  by  a  small 
cut,  made  with  antiseptic  precautions,  and  the  mate- 
rial introduced  through  a  fine  opening  into  it  by 
means  of  a  syringe. 

Reception  of  Bacteria  with  the  Food. — Feeding 
Experiments. — In  the  artificial  infection  by  feeding, 
the  possible  injury  of  the  mucous  membrane  which 
may  be  produced  by  rough,  prickly  food,  must  be 
avoided.  Infection  through  such  an  eroded  spot  is  to 
be  viewed  as  a  wound-infection  in  its  entire  effects, 
and  regarded  as  having  a  different  significance,  as  to 
the  aetiology  of  the  spontaneous  appearance  of  the 
disease,  from  the  reception  of  infectious  material 
through  the  food  without  erosion  of  the  mucous  mem- 
brane. 

According  to  Koch,  Gaffky,  and  L6fi1er,J  this  ob- 
ject is  most  certainly  attained,  at  least  in  the  larger 
animals,  in  the  following  manner :  a  thin  layer  is  so 
far  cut  from  a  small,  fresh  rectangular  piece  of  po- 
tato that  it  can  be  lifted  up  like  a  cover,  and  then 
the  interior  is  hollowed  out  and  filled  with  the  bac- 
teria-material in  pure  culture.  The  cover  is  replaced, 
and  the  piece  of  potato  thus  prepared  is  placed  upon 

*  "  Wiener  Hied.  Jahrbucher,"  1883. 

t  "  Beitrag  zur  Impftuberkulose."     "  Deutsche    med.  "Wochen- 
schrift,"  1883,  No.  36. 

\  "  Mittheilungen  aus  dem  kaiserl.  Gesundheitsamte,"  1884,  Bd. 
II,  S.  166. 

12 


176  BACTERIOLOGICAL  INVESTIGATION. 

the  back  part  of  the  tongue,  so  that  it  must  be  swal- 
lowed unmasticated.  Small  cubes  of  hard  -  cooked 
white  of  egg  may  be  prepared  in  the  same  way.  The 
bacteria  -  material  then,  to  the  exclusion  of  every 
abrasion  of  the  mucous  membrane,  passes  into  the 
stomach.  The  previous  experiments  show  that  fresh 
spore-free  bacteria,  as  a  rule,  are  destroyed  by  the 
acid  of  the  gastric  juice,  and  do  not  produce  infec- 
tion. On  this  account  experiments  should  be  made 
with  both  spore-free  and  spore- containing  material. 

Since  infection  seems  to  take  place  more  easily  in 
gastro-intestinal  catarrh  (at  least,  according  to  experi- 
ments, a  favorable  influence  on  the  infection  may  be 
brought  about  by  all  conditions  which  directly  or  in- 
directly interfere  with  the  acidity  of  the  stomach), 
the  deleterious  influence  of  the  gastric  juice  may  be 
avoided  in  experiments  with  bacteria  which  form  no 
spores,  by  producing  artificially  a  gastro-intestinal  ca- 
tarrh by  drastic  cathartics  before  feeding  the  animal 
with  the  material  containing  bacteria  ;  or,  according 
to  Mcati  and  Bietsch,*  the  bacteria  may  be  injected 
directly  into  the  duodenum.  This  operation  is  done, 
under  antiseptic  precautions,  with  sterilized  instru- 
ments, and  with  cotton  and  silk  sterilized  by  steam. 
The  animal,  after  being  tied,  is  covered  with  a  layer 
of  rubber  tissue  sterilized  by  steam.  This  tissue  has 
an  opening  only  over  the  spot  for  the  operation.  The 
skin  of  the  abdomen  seen  through  this  opening  is 
freed  from  hair  and  washed  with  soap  and  water  over 
its  entire  extent,  then  thoroughly  washed  with  a  1  per 
mille  solution  of  sublimate,  and  finally  the  sublimate 
is  removed  by  sterilized  water.  The  incision  is  made 
in  the  linea  alba  somewhat  below  the  xiphoid  carti- 

*  u  Semaine  m6d.,"  1884,  No.  38.    Vergl.  auch  Deneke,"'"  Deutsche 
med.  Wochenschrift,"  1885,  No.  3. 


PARASITIC  BACTERIA.  177 

lage.  After  opening  the  peritonaeum,  the  pyloric  end 
of  the  stomach  is  found  as  quickly  as  possible  and  is 
followed  along  in  the  mesentery  to  the  duodenum. 
This  is  then  fixed  with  pincettes,  and  the  needle  of  the 
sterilized  syringe  (Pravaz  syringe,  as  modified  by 
Koch)  is  introduced  in  the  direction  of  the  long  di- 
ameter of  the  gut,  and  the  contents  of  the  syringe 
so  injected  that  nothing  passes  into  the  abdominal 
cavity.  (Concerning  the  construction  of  the  syringe, 
cfr.  Subcutaneous  Injections.)  Then  the  duodenum 
is  replaced  in  the  abdominal  cavity.  The  wound  is 
united  by  tight  sutures  and  sprinkled  with  iodoform. 
Any  further  dressing  is  superfluous. 

Since  infection  is  possible  not  only  by  the  bacteria 
taken  in  with  the  solid  food,  but  also  by  fluid-nutri- 
ment, as  water  and  milk,  it  is  in  many  cases  better  to 
introduce  the  pure  cultures  in  sterilized  water  or  milk 
which  is  used  for  drinking. 

Infection  brought  about  through  abrasions  is  ex- 
perimentally more  at  command  than  that  produced 
by  means  of  the  air  and  food.  This  method,  strict- 
ly, ought  to  be  used  only  for  the  genuine  traumatic 
infectious  diseases.  On  account  of  its  certainty  and 
availability  it  has  furnished  a  quite  general  means  for 
the  decision  of  many  pathological  questions  where 
the  mode  of  the  spontaneous  infection  is  not  exactly 
known. 

The  Cutaneous  Inoculation.  —  Cutaneous  inocu- 
lation consists  in  producing  an  abrasion  of  the  skin, 
without  injury  to  the  subcutaneous  tissue,  with  a 
subsequent  or  simultaneous  application  of  the  infect- 
ing material.  A  slight  abrasion  is  made  with  a  steril- 
ized knife  on  the  skin  denuded  of  hair,  and  upon  this 
superficial  wound  a  particle  of  the  pure  culture  or  of 
a  fluid  containing  bacteria  is  placed  with  a  knife  or 


178  BACTERIOLOGICAL  INVESTIGATION. 

platinum  wire.  In  place  of  this  operation,  performed 
at  two  different  times,  the  knife  can  also  be  dipped 
into  the  pure  culture  and  the  incision  made  directly 
with  the  infected  knife. 

Spots  are  chosen  where  the  animal  can  not  lick 
the  wound,  and  which,  in  addition,  permit  the  course 
of  the  process  to  be  followed  as  much  as  possible. 
The  ear  is  especially  suitable  for  this.  A  cut  not  draw- 
ing blood,  or  only  a  few  millimetres  deep,  is  made  on 
the  inner  surface  of  the  ear,  not  far  from  the  point 
or  on  the  anterior  root  in  the  free  skin  bordering  on 
the  cartilage-rim.  In  mice,  a  good  cutaneous  abrasion 
can  be  made  almost  only  at  the  root  of  the  ear,  and 
even  there  it  is  difficult. 

The  inoculations  in  the  transparent  epidermis, 
which  were  made  by  Nassilauf  *  and  Eberth,f  can  be 
included  here.  A  superficial  puncture  is  made  with 
an  infected  needle  in  the  transparent  epidermis,  and 
from  this  puncture  bacteria  develop  in  a  star- shaped 
form — the  so-called  fungous  figures. 

Subcutaneous  Applications. — The  subcutaneous 
application  of  bacteria  is  to  be  distinguished  from  the 
true  cutaneous  inoculation.  In  a  spot  denuded  of 
hair,  where  it  can  not  be  licked,  a  fold  of  the  skin  is 
raised  with  the  pincettes  and  incised.  With  a  knife  or 
blunt  probe  a  pocket  is  made  under  the  skin.  In  this 
pocket,  with  a  sterilized  knife  or  platinum  needle,  a 
particle  from  a  pure  culture  is  placed,  and  then  the 
skin  is  again  pressed  down.  In  mice,  according  to 
Koch,  J  the  operation  is  most  conveniently  done  by 
seizing  the  tail  of  the  mouse  with  long  pincettes  while 

*  Yirchow's  "  Archiv,"  Bd.  L,  8.  550. 
t  "Bakterische  Mykosen,"  1872. 

\  "  Die  Aetiologie  der  Milzbrandkrankheit."  "  Beitrfige  zur 
Biologie  der  Pflanzen,"  Bd.  II,  2.  Heft,  1876,  S.  279. 


PARASITIC  BACTERIA.  179 

it  is  sitting  in  a  large  glass  cylinder  that  is  covered, 
and  drawing  the  tail  out  of  a  space  between  the  cover 
and  glass  rim  not  large  enough  for  the  mouse  to  pass, 
so  that  it  can  be  well  grasped  in  the  left  hand.  Then, 
at  the  root  of  the  tail,  in  the  loose  skin,  a  superficial 
cross-cut  is  made  with  the  scissors,  and  then  a  pocket, 
in  which  a  particle  of  the  pure  culture  is  placed  with 
a  knife. 

Subcutaneous  Injections. — For  subcutaneous  in- 
jections a  Pravaz  syringe,  as  modified  by  Koch,*  is 
used,  which  can  be  thoroughly  sterilized  by  heat. 
For  this  reason  it  is  made  without  rubber.  The 
metal  extremities  are  united  to  the  glass  cylinder 
by  a  screw  ground  in  the  glass,  and  the  joint  is 
made  close  by  a  small  cork  washer,  which  is  often  re- 
newed. The  piston  of  the  syringe  is  wound  each  time 
with  fresh  cotton  and  silk  thread  until  it  fits  close- 
ly. The  syringe,  thus  prepared,  is  kept  for  two 
hours  in  a  dry-oven  at  a  temperature  of  150°  to 
160°  C.,  and,  after  cooling,  is  protected  from  dust. 
Before  use,  the  piston  is  moistened  in  fresh  sterilized, 
distilled  water.  For  injection,  the  pure  cultures  of 
bacteria,  suspended  in  sterilized  water,  freshly  pre- 
pared, are  employed. 

The  introduction  of  the  bacteria  into  the  anterior 
chamber  of  the  eye  is  also  of  importance,  especially 
in  dealing  with  forms  which  develop  so  slowly  that 
the  first  visible  effects  present  themselves  in  the  iris 
after  all  the  evidences  of  inflammation  have  subsided, 
Cohnheim  and  Salomonsen  f  introduced  these  meth- 


*  u 


;Mittheilungen,"  I,  1881,  S.  17. 
t  "Sitzungsberichte  der  schlesischen  Gesellschaft    f&r  vaterlan- 
dische  Cultur  vom  13.  Juli,  1877";  und  Salomonsen,  "  Om  Indpod- 
ning  af  Tuberkulose  sarligt  i  Kaninens  Iris."     "  Nordiskt  Medicinskt 
Arkiv,"  1879,  Bd.  XI,  Nos.  12  u.  19. 


180  BACTERIOLOGICAL  INVESTIGATION. 

ods  for  tuberculosis.  The  technique  is  the  same  as  in 
iridectomy.  By  means  of  fixation-pincettes  held  in 
the  left  hand,  a  fold  of  the  conjunctiva  is  seized  and 
drawn  to  the  side  of  the  pupil.  The  other  hand  intro- 
duces, close  to  the  edge  of  the  cornea,  a  lance,  direct- 
ed obliquely  toward  the  interior.  As  soon  as  the 
point  reaches  the  anterior  chamber,  the  handle  is 
inclined  so  that  the  point  is  directed  against  the  cor- 
nea. In  this  manner  also  must  the  knife  be  with- 
drawn, as  soon  as  the  incision  is  large  enough,  so 
that  the  iris  and  lens  may  not  be  injured  as  they  press 
forward,  on  account  of  the  escape  of  the  fluid  from 
the  anterior  chamber.  Pure  cultures  can  then  be 
thrown  in  with  the  syringe,  while  small  particles  of 
material  containing  bacteria  can  be  introduced  with 
the  iris-pincettes. 

Direct  Injection  into  the  Circulation. — For  the 
direct  injection  into  the  circulation  the  vessel  (usually 
the  jugular  or  crural  vein)  is  exposed  with  antiseptic 
precautions,  and  the  needle  of  the  Koch  syringe  is  fast- 
ened in  the  same  way  as  the  capillary  tube  of  Salo- 
monsen  (page  123),  but  so  that  as  little  blood  as  possi- 
ble flows  away.  The  ligation  of  the  injured  vessel, 
and  also  the  dressing  of  the  wound  in  the  skin,  is 
done  in  the  ordinary  way,  as  in  surgery. 

If  it  is  not  desired,  or  is  impossible,  to  make  in- 
oculations with  pure  cultures,  but  the  inoculation 
is  to  be  made  from  animal  to  animal,  the  technique 
is  still  the  same,  except  that  in  obtaining  material  it 
must  be  as  carefully  done,  just  as  if  it  were  desired 
to  use  it  for  the  preparation  of  pure  cultures  in  blood- 
serum. 

If  the  virulent  peculiarities  of  a  form  of  bacteria 
have  been  determined  in  pure  cultures,  it  is  desirable 
to  know  approximately  the  smallest  number  of  the 


PARASITIC  BACTERIA.  181 

bacteria  which  will  bring  about  certain  infection  ;  or 
if  the  virulence  of  an  infectious  fluid  is  known,  the 
blood,  for  example,  it  is  desirable  to  know  in  what 
degree  of  dilution  a  certain  number  of  drops  will 
surely  produce  infection.  From  such  experiments 
an  almost  direct  conclusion  may  be  drawn  as  to  the 
mode  of  the  natural  infection.  As  yet  only  quite 
general  principles  in  this  direction  have  been  deter- 
mined, which  show,  according  to  Koch,  that  the 
minimum  number  of  bacteria  absolutely  essential  for 
infection  fluctuates,  and  that  this  minimum  is  in 
relation  to  the  body-weight  of  the  animal.  The  cal- 
culation corresponding  to  the  proposition  has  been 
determined  for  only  one  form  of  septicaemia  by  Da- 
vaine,*  Koch,f  and  Gaffky.  f 

Bacteria  have  as  yet  won  but  little  significance  in 
the  pathology  of  plants,  perhaps  on  account  of  the 
general  acid  reaction  of  vegetable  tissue.  Reinke  and 
Berthold  *  succeeded  in  infecting  a  sound  potato  with 
a  fluid  containing  bacteria  from  a  damp,  decomposing 
potato.  Since  the  intact  skin  of  a  sound  potato  pre- 
vents infection,  this  must  be  cut  through.  For  this 
purpose  a  three-sided  pyramid  is  cut  out  with  a  knife, 
and  into  this  wound  a  drop -of  the  fluid  or  a  particle 
of  the  pure  culture  is  introduced,  and  the  removed 
piece  is  replaced.  The  inoculated  tuber  is  then 
placed  in  a  moist  chamber.  According  to  Wakker,  || 

*  "  Bulletin  de  1'academie  de  medicine,"  Sitzung  vom  17.  Sep- 
tember, 1872. 

t  "  Untersuchungen  fiber  die  Aetiologie  der  Wundinfectionskrank- 
heiten,"  1878. 

t  "  Experimentelle  erzeugte  Septicaemie  mit  Rucksicht  auf  progres- 
sive Virulenz  und  accommodative  Zuchtung."  "  Mittheilungen  a.  d. 
kais.  Gesundheitsamte,"  Bd.  I,  1881,  S.  60. 

*  "  Unters.  aus  dem  botan.  Laborat.  in  Gottingen,"  Heft  I. 
I  "Botan.  Centralblatt,"  Bd.  XIV,  S.  315. 


182          BACTERIOLOGICAL  INVESTIGATION. 

the  so-called  yellow  disease  of  the  hyacinth,  observed 
in  Holland,  is  produced  by  bacteria  which  appear  as 
yellow,  slimy  masses  in  the  vessels  of  the  bulb,  and 
in  the  vessels  and  parenchyma  of  the  leaves.  The 
direct  proof,  that  these  bacteria  were  the  cause  of  the 
disease,  was  ascertained  by  transfers  to  sound  plants. 


V. 

GENERAL  BIOLOGICAL  PROBLEMS. 

AFTER  it  has  been  shown  by  inoculation  that  the 
bacteria  observed  in  a  decomposition  or  disease  are 
the  cause  of  this  process,  it  must  be  determined 
whether  a  septic  form  produces  only  this  one  decom- 
position or  fermentation,  or  whether  it  acts  differently 
in  other  media.  Thus,  e.  g.,  the  bacilli  of  the  butyric- 
acid  fermentation  produce  this  form  of  fermentation 
in  saccharine  substances,  while  in  glycerine,  according 
to  Fitz,  they  form  propyl  alcohol  as  the  chief  product. 

Further,  it  is  to  be  noted  whether  ferment  bacteria 
produce  characteristic  pigment  on  certain  substances, 
or  whether  they  may  act  as  pathogenic  micro-organ- 
isms upon  certain  animals.  According  to  Brieger, 
e.  g.,  certain  bacilli,  which  produce  proprionic-acid 
fermentation  in  saccharine  substances,  kill  guinea- 
pigs.  On  this  account  it  is  to  be  determined,  by  the 
greatest  possible  variation  in  the  nutrient  media  and 
by  the  inoculation  of  different  species  of  animals, 
whether  the  septic  bacteria,  and  especially  the  fer- 
ment and  pigment  bacteria,  produce  only  one  single 
decomposition,  or  whether  they  produce  different  de- 
compositions in  different  substances ;  whether  they 
form  pigment,  and  whether  they  are  capable  of  a 
parasitic  form  of  life.* 

*  Ofr.  iiber  diese  Moglichkeiten :  Fitz,  "  Berichte  der  deutschen 


184  BACTERIOLOGICAL  INVESTIGATION. 

In  the  case  of  parasitic  bacteria  it  is  to  be  deter- 
mined whether  they  possess  virulent  peculiarities 
only  for  the  one  species  of  animal  in  which  they  are 
spontaneously  developed,  or  whether  they  are  also 
pathogenic  for  other  animals.  The  reply  to  this 
question  is  of  the  greatest  importance  for  the  deter- 
mination of  the  possibility  of  the  extension  of  an  in- 
fectious disease.  The  pure  cultures  of  the  infectious 
bacteria  are  to  be  transferred  to  different  media,  since 
they  may  possibly  decompose  a  substance  in  a  more 
typical  manner  upon^which  they  are  able  to  live. 
The  bacilli  of  glanders  and  the  vibriones  of  cholera 
Asiatica,  e.  g.,  produce  in  certain  media  a  brown 
pigment,  and  the  cocci  of  osteo-myelitis  call  forth 
lactic-acid  fermentation  and  produce  a  yellow  pig- 
ment. 

The  preparation  of  the  media,  and  the  transfers  to 
these  and  to  animals,  is  accomplished  in  the  manner 
previously  described.  Here  it  is  especially  to  be 
noted  whether  with  the  change  of  the  medium  the 
form  is  altered  in  any  way,  or  whether  new  forms 
develop.  In  the  preparation  of  pure  cultures  a  care- 
ful control  of  the  inoculation  is  to  be  made  as  soon  as 
any  such  peculiarities  are  observed. 

With  the  aid  of  pure  cultures  it  is  to  be  deter- 
mined whether  the  bacteria  are  able  to  free  the  nitro- 
gen necessary  for  their  growth  only  from  albumi- 
nates,  and  the  carbonic  acid  only  from  sugar,  or 
whether  they  can  take  it  up  in  a  simpler  union.  The 
compounding  of  such  solutions  and  the  variations  of 
the  mineral  constituents  follow  the  rule  given  in  the 
formulae  (Section  III,  page  94). 

Are  the  bacteria  able  to  peptonize  solid  albumen 

chemischen  Gesellschaft,"  1882,  S.  867;  1884,8.1188.    Und  meine 
Ausgaben  in  der  "Deutsche  med.  Wochenschrift,"  1884,  No.  48  bis 50. 


GENERAL  BIOLOGICAL  PROBLEMS.  185 

and  hard-cooked  fibrin,  or  to  dissolve  coagulated 
casein  ? 

Casein,  freshly  precipitated  by  acid,  is  washed 
with  distilled  water,  to  remove  the  acid  reaction,  and 
in  the  same  way  freshly  prepared  fibrin  is  thoroughly 
washed.  It  is  best  to  make  three  parallel  experi- 
ments, in  which,  in  some  test-tubes,  only  a  piece  of 
casein,  a  flake  of  fibrin,  or  a  cube  of  hard-cooked 
white  of  egg  is  introduced  with  distilled  water.  In 
other  tubes  O'l  per  cent  of  beef -extract  is  added,  and 
in  a  third  series  of  tubes,  in  addition,  0*5  per  cent  of 
grape-sugar  is  also  added.  The  test-tubes  are  first 
thoroughly  sterilized  by  steam,  and,  after  cooling, 
inoculated,  and  some  are  kept  at  the  temperature  of 
the  room  and  others  in  a  culture-oven.  Finally,  the 
question  of  anaerobiosis  is  to  be  taken  into  consider- 
ation. 

By  the  inoculation  of  sterilized  milk  it  is  to  be  de- 
termined whether  bacteria  produce  coagulation  of  the 
casein  by  the  formation  of  acid,  or  whether  they 
separate  the  casein  and  then  dissolve  it  after  separa- 
tion. 

By  the  inoculation  of  milk,  sugar,  or  cane-sugar, 
it  is  found  that  the  bacteria  make  use  of  double  sac- 
charates.  Wherefore  it  is  to  be  noted  whether  these 
double  saccharates  are  resolved  into  the  simple  sac- 
charates.  By  transfers  to  solutions  of  starch  and  to 
starch-paste  it  is  to  be  determined  whether  the  bacteria 
dissolve  starch  and  convert  it  into  sugar  or  not.  For 
these  experiments,  in  some  test-tubes  nothing  is  added 
to  the  sugar  or  the  starch,  in  others  1  per  cent  of  beef- 
extract,  and  in  a  third  series  1  per  cent  of  beef-ex- 
tract and  5  per  cent  of  peptone  are  added. 

Enzyme. — All  such  hydrolytic  divisions  as  are 
produced  by  diastase,  pepsin,  rennet,  etc.,  are,  with 


186  BACTERIOLOGICAL  INVESTIGATION. 

tolerable  certainty,  reduced  by  the  higher  organisms 
to  the  chemical  ferment  enzyme,  which  can  be  sepa- 
rated from  the  cells  producing  the  same,  and  then 
produce  hydration  outside  of  the  organism  also.  If 
such  hydration  is  produced  by  bacteria,  it  is  to  be 
determined  directly  by  experiment  whether  these 
processes,  separable  from  the  bacteria,  are  real  en- 
zyme productions,  or  whether  they  can  not  be  sepa- 
rated from  the  living  cells.  Hydration  *  without 
the  presence  of  a  separable  enzyme  was  certainly  de- 
termined in  the  lower  animals  by  Krukenberg.  I 
was  not  able  to  separate  the  hydration  of  milk-sugar 
by  the  lactic-acid  bacilli  from  these  bacteria,  and  a 
recent  account  by  Hansen  of  a  direct  alcoholic  fer- 
mentation of  cane-sugar  produced  by  a  mould  fun- 
gus without  the  separation  of  invertin  is  in  this 
sense  also  significant.  There  is  not  the  slightest 
ground  for  supposing  that  every  hydration  brought 
about  by  bacteria  is  without  a  separable,  isolable, 
true  enzyme  as  the  active  agent ;  but  concerning 
this  only  direct  experiments  are  decisive.  The  de- 
tails of  these  facts  belong  essentially  to  the  domain 
of  chemistry,  f  The  general  process  is  that  the  en- 
zyme, with  the  albuminates,  is  precipitated  by  alco- 
hol ;  these  are  taken  up  with  glycerine  or  water,  and 
in  this  way  separated  from  the  albuminates,  and 
this  is  finally  repeated.  These  procedures  must  be 
carried  on  in  sterilized  solutions  which  have  been 
inoculated  with  pure  cultures,  and  at  a  time  when 
as  yet  no  spores  are  present.  Weigert  %  had  already 

*  Kuhne,  "  Erfahrungen  und  Bemerkungen  fiber  Enzyme  und  Fer- 
mente."  "  Untersuch.  des  physiologischen  Instituts  der  Universitat 
Heidelberg,"  Bd.  I,  Heft  3,  1877. 

t  Ofr.  A.  Mayer,  "DieLehre  von  den  chemischen  Fermenten," 
1882. 

{  '*  Ueber  Glycerin  als  Unterscheidungsmittel  geformter  und  un- 


GENERAL  BIOLOGICAL  PROBLEMS.  187 

previously  found  that  glycerine*  for  this  purpose 
sometimes  failed,  and  I  found  that  alcohol  and  gly- 
cerine were  not  always  reliable  for  the  separation  of 
the  enzyme,  especially  if  spores  are  present. 

The  separation  of  the  enzyme  from  fluid  contain- 
ing bacteria  may  be  also  attempted  by  filtration.  In 
the  experiments  concerning  enzyme  it  is  absolutely 
necessary  to  determine,  by  controlling  pure  cultures, 
especially  plate-cultures,  whether  bacteria,  in  spite 
of  all  care,  were  not  also  transferred. 

Ptomaines. — While  with  the  parasitic  bacteria, 
in  the  narrowest  sense,  the  transfer  of  the  infection 
is  closely  united  to  the  transfer  of  the  bacteria,  still 
the  action  as  such,  perhaps,  may  be  separable  through 
this  fact,  that  these  bacteria  form  an  insoluble  en- 
zyme or  a  basic  body  similar  to  an  alkaloid — viz., 
ptomaine — which  produces  the  peculiar  poisonous 
action.  By  the  formation  of  such  poisonous  products 
from  the  albuminates  the  putrefactive  bacteria  them- 
selves may  produce  intoxication  without  bringing 
about  a  genuine  infectious  disease.  The  methods  of 
analyses  for  such  poisons  are  in  part  the  same  as  with 
other  enzymes  ;  in  part  related  to  the  method  of  the 
determination  of  the  alkaloids  according  to  the  pro- 
cedure of  Stas-Otto  ;  f  in  part,  methods  are  yet  to  be 
worked  out,  as  has  recently  been  done  by  Brieger4 

While  a  greater  part  of  the  previously  described 
biological  problems  can  only  be  solved  by  the  chemi- 

geformter  Fermente."  "  Deutsche  med.  "Wochenschrift,"  1877,  Nos. 
40,  41. 

*  "  Mittheilungen  aus  dem  k.  Gesundheitsamte,"  Bd.  II,  1884,  S. 
843. 

t  Otto,  "  Anleitung  zur  Ausmittlung  der  Gifte,"  4te  Aufl.,  1884. 
Ludvig,  "Medizinische  Ohemie,"  1885. 

\  "Ueber  Ptomaine,"  1885. 


188 


BACTERIOLOGICAL  INVESTIGATION. 


cally  educated  observer,  yet  a  few  fundamental  ques- 
tions remain  of  an  essentially  experimental  character. 

BEHAVIOB  TO  TEMPERATURES. 

It  is  to  be  determined  what  is  the  lowest  tempera- 
ture at  which  a  purely  cultivated  bacterium  begins  to 
multiply  and  develop  into  colonies.  For  this  pur- 
pose gelatin  in  test-tubes  and  sterilized  solutions  are 
most  advantageously  inoculated  with  pure  cultures, 
and  placed  for  a  week  in  an  ice-chest,  the  tempera- 
ture of  which  in  general  does  not  fall  below  5°  C. 
nor  rise  above  8°  C.  Temperatures  from  8°  C.  to  the 
temperature  of  the  room  are  best  attained  in  cool 

rooms,  in  vessels  with  dou- 
ble walls,  through  which  cold 
hydrant  -  water  continually 
circulates ;  for  temperatures 
from  15°  C.  to  blood  temper- 
ature, by  the  insertion  of  a 
gas -pressure  regulator,  the 
ordinary  culture-ovens  can 
be  used. 

For  most  cases  the  ther- 
mostat, according  to  d'Ar- 
sonval  (Fig.  28),  is  recom- 
mended for  very  exact  ob- 
servations. This  consists  of 
a  double  -  walled  cylinder 
made  of  strong  sheet-cop- 
per, which  ends  below  in  a 
double-walled  conical  heating  surface.  The  inclined 
portion  of  the  cylinder  from  M'  to  ^permits  the  re- 
moval of  all  air  and  the  complete  filling  of  the  en- 
tire mantle  ( W)  with  water.  The  mantle  is  complete- 
ly filled  to  M'  with  distilled  or  rain  wafer  at  a  tern- 


GENERAL  BIOLOGICAL  PROBLEMS.  189 

perature  approximately  near  the  desired  degree. 
Then  the  standing-pipe  (S)  is  introduced,  in  which 
the  water  rises  to  a  certain  height.  The  gas,  independ- 
ent of  the  pressure  in  the  remaining  pipes,  passes 
through  a  pressure-regulator,  as  follows :  at  a  it  en- 
ters through  an  opening  (a')  into  the  small  chamber 
(7i) ;  from  here  it  goes  out  again  at  &,  and  passes  from 
&  through  a  tube  uniting  it  to  c,  and  then  to  the 
burners  (d).  These  flames  are  protected  against  the 
draught  by  chimneys  of  isinglass  and  can  not  be 
extinguished.  Between  the  room  (7i)  and  the  water- 
mantle  ( W)  a  Schloesing's  membrane-regulator  (mm) 
is  extended.  In  the  temperature  oscillations  of  the 
water  in  the  mantle  ( W)  the  contraction  or  expansion 
of  the  large  mass  of  water  makes  itself  visible  by 
the  relatively  greater  fall  or  rise  of  the  water  present 
in  the  narrow  standing-pipe  (S).  In  this  way  the 
pressure,  relatively  great,  that  is  resting  upon  the 
water  is  altered,  and  the  rubber  membrane  (mm)  is 
correspondingly  drawn,  sometimes  more  toward  the 
water,  at  others  pressed  closer  to  the  escape- opening 
of  the  gas  (a').  More  gas  proportionately  passes  at 
times  through  a'  into  the  room,  sometimes  less,  and, 
as  a  result  of  this,  the  flames  burn  sometimes  higher 
and  again  lower,  till  the  temperature  is  reached  at 
which  the  apparatus  was  set. 

The  regulation  to  one  tenth  of  a  degree  is  ex- 
act for  several  weeks,  if  the  height  of  the  water 
in  the  standing-tube  is  daily  controlled  and  is  regu- 
lated by  removing  a  few  drops  or  by  adding  a  few 
drops  of  distilled  water,  and  if  the  gas-pressure 
is  independent  of  the  gas-pressure  of  the  general 
supply,  and  the  room  in  which  the  apparatus 
stands  has  an  equal  temperature.  In  case  of  greater 
temperature  oscillations  of  the  room,  it  is  neces- 


\ 
190  BACTERIOLOGICAL  INVESTIGATION. 

sary  to  surround  the  apparatus  with  a  mantle  of 
asbestos. 

With  the  aid  of  a  thermostat  the  most  favorable 
temperature  can  be  determined — i.  e.,  that  tempera- 
ture at  which  the  bacteria  multiply  most  rapidly  and 
act  most  intensely.  For  this  purpose,  nutrient  gela- 
tin, agar-agar,  or  blood-serum  is  inoculated  with  a 
pure  culture  and  the  time  is  determined  within  which 
distinctly  characteristic  colonies  appear.  Then  steril- 
ized nutrient  solutions  in  test-tubes  and  Erlenmeyer's 
flasks  are  inoculated  with  pure  cultures,  and  it  is  de- 
termined by  tests,  made  from  time  to  time,  at  what 
temperature  the  maximum  of  the  action — e.  g. ,  a  cer- 
tain degree  of  acidity — is  attained  in  the  shortest  time. 
The  temperature  most  favorable  for  multiplication 
and  action  is  restricted  sometimes  within  a  few  de- 
grees, but  often  also  shows  great  latitude.  Beyond 
this  point  ensues  again  a  diminution  of  the  rapidity 
of  the  growth  and  intensity  of  the  action.  For  the 
determination  of  these  limits,  pure  cultures  are  inocu- 
lated both  in  solid  media  and  in  favorable  fluids — 
e.  g.,  thus  it  is  determined  that  the  multiplication 
and  the  action  of  the  lactic-acid  bacteria  cease  be- 
tween 45-3°  C.  and  45 '5°  C.  in  milk  and  3  to  4  per 
cent  sugar  solutions,  without  the  death  of  the  bac- 
teria ensuing.  These  limits  indicate  at  first  only 
a  cessation  of  development,  a  kind  of  fixation  by 
heat.  Beyond  these  the  first  real  destruction  of  the 
bacteria  ensues. 

After  Pasteur,  in  February,  1880,  had  made  the 
discovery  that  by  biological  interference  the  virulence 
of  chicken-cholera  was  diminished,  and  that  inocula- 
tion with  this  attenuated  virus  protected  the  animals 
against  inoculation  with  virulent  material,  Toussaint 
found  that  virulent  anthrax-blood,  by  warming  ten 


GENERAL  BIOLOGICAL  PROBLEMS.  191 

minutes  at  55°  C.,  or  by  the  addition  of  carbolic  acid, 
was  rendered  less  virulent,  and  Pasteur,  a  little  later, 
found  that  by  cultivating  the  pure  anthrax-bacilli  in 
bouillon  at  42°  to  43°  C.  the  virulence  of  this  bacte- 
rium could  be  systematically  attenuated  ;  then  Chau- 
veau*  discovered  that  at  52°  C.  fifteen  minutes,  at 
50°  C.  twenty  minutes,  at  47°  C.  three  or  four  hours, 
at  43°  C.  about  six  days,  and  at  42°  C.  nearly  twenty 
days  were  necessary  to  obtain  the  same  degree  of  at- 
tenuation, but  that  the  certainty  and  the  constancy 
of  attenuation  were  the  greater  the  lower  the  tem- 
perature. Koch,  Gaffky,  and  Loffler  f  made  these  ob- 
servations for  the  temperature  between  42°  and  43°  C. 
still  more  precise  and  exact. 

Such  fine  gradations  of  the  temperature  can  only 
be  obtained  with  an  excellent  thermostat.  In  a 
similar  manner  it  is  to  be  determined  at  what  high 
or  low  temperatures  spores  form,  and  below  and 
above  what  temperatures  no  spores  are  formed. 
Within  these  limits  it  is  to  be  determined  what  is 
the  most  favorable  temperature  for  the  formation  of 
spores,  so  that  the  number  of  the  spores  and  the 
regularity  of  their  formation  may  be  observed  in 
cultures,  and  the  time  ascertained  at  which  the  first 
spores  appear.  Also  the  time  necessary  for  the  ger- 
mination is  to  be  determined  at  different  degrees  of 
temperature.  These  deductions  concerning  the  low- 
est temperature  at  which  a  form  of  bacteria  may  still 
multiply,  concerning  the  height  and  extent  of  the 
most  favorable  temperature,  and  concerning  the  high 
temperature  at  which  a  form  does  no  longer  act; 
further,  the  observations  concerning  the  high  and  low 

*  "Oomptes  rendus,"  T.  96,  1883,  Nos.  9,  10,  11. 
t  "Mittheilungen  aus  dem  k.  Gesundheitsamte,"  Bd.  II,  1884,  S. 
147. 

13 


192          BACTERIOLOGICAL  INVESTIGATION. 

temperatures  at  which  the  formation  of  spores  no 
longer  occurs,  and  the  deduction  concerning  the  most 
favorable  temperature  for  spore-formation,  and  the 
temperature  necessary  for  spore-germination — almost 
alone  permit  the  correct  conclusion  as  to  the  degree 
of  the  adaptability  to  the  parasitic  mode  of  life. 

If  the  difficulty  or  the  ease  of  the  preparation  of 
the  pure  cultures  is  taken  into  consideration  ;  if  it  is 
noted  whether  the  purely  cultivated  bacteria,  in  re- 
spect to  their  need  of  nitrogen  and  carbonic  acid,  are 
very  dainty  or  not ;  if  the  favorable  conditions  of 
temperature  described  are  tested — then  the  pure  cult- 
ure is  under  good  conditions  for  an  epidemiological 
experiment.  The  experimenter  can  by  these  inqui- 
ries protect  himself  against  the  one-sided,  contagious 
significance  of  the  interpretation  of  the  experiments 
on  animals,  and  can  come  unprejudiced  to  the  review 
of  the  local  and  temporary  auxiliary  causes  for  the 
origin  of  an  epidemic  or  endemic  disease. 

In  order  to  determine  more  exactly  the  temperature 
at  which  spores  are  killed,  it  is  best  to  proceed  by  using 
a  deep  water-bath,  which  has  a  constant  level  of  the 
water.  Upon  the  bottom  of  the  water-bath  a  wooden 
plate  is  placed  to  protect  against  the  direct  action  of 
the  hot  bottom.  Above,  the  bath  is  closed  with  a  cover 
which  has  a  number  of  openings  for  the  introduction 
of  several  thermometers,  extending  to  different  depths, 
and  for  the  reception  of  test-tubes  containing  sterilized 
nutrient  solutions.  The  water  of  the  water-bath  must 
stand  higher  than  the  plane  of  the  fluid  in  the  tubes, 
in  which  the  temperature  remains  somewhat  lower 
than  that  in  the  water-bath,  so  that  the  indicated  de- 
gree does  not  correspond  exactly  to  the  temperature 
really  obtained  in  the  test-tubes.  When  an  equaliza- 
tion of  the  temperature  has  been  obtained,  after  about 


GENERAL  BIOLOGICAL  PROBLEMS.  193 

half  an  hour,  the  tubes,  having  been  opened,  are  rap- 
idly inoculated  with  spores,  and  repeatedly  moved  to 
and  fro.  The  tubes  are  left  at  different  temperatures 
— e.  g.,  boiling  temperature,  95°  C.,  and  from  90°  to 
70°  C.  for  different  lengths  of  time  ;  then  a  few  test- 
tubes  are  placed  at  the  temperature  most  favorable 
to  the  development  of  the  form  under  consideration, 
in  order  that  it  may  be  determined,  by  the  appearance 
or  non-appearance  of  cloudiness,  as  to  the  death  of  the 
germs.  Gelatin  may  be  mingled  with  the  contents  of 
other  tubes,  and  these  used  for  plate-cultures.  In  the 
study  of  the  effects  of  temperature  above  100°  C.,  the 
salt-  or  oil-bath  is  best  used  for  the  experiments. 

The  spores,  also,  before  being  killed,  may  be  at- 
tenuated, as  is  shown  by  transfers  to  suitable  solu- 
tions or  animals.  In  order  to  determine  the  influ- 
ence of  dry  heat,  short  pieces  of  silk  thread  are 
soaked  in  a  fluid  free  from  spores,  and  in  another 
containing  spores,  by  laying  the  threads  in  a  suspen- 
sion of  a  pure  culture  of  the  corresponding  bacteria 
in  distilled  water,  or  by  placing  them  in  fresh  blood 
containing  bacteria,  or  in  the  tissue-fluid.  Then  the 
threads  are  dried  in  an  exsiccator,  laid  in  a  watch- 
glass,  and  directly  exposed  in  a  dry-oven  to  differ- 
ent elevations  of  temperature. 

According  to  the  kind  of  action  of  the  tempera- 
ture, as  mentioned,  a  simple  checking  of  development, 
or  an  attenuation  of  virulence  or  destruction  of  the 
bacteria,  is  made  perceptible. 

Besides  temperature,  many  agents  act  according 
to  concentration  in  preventing  development,  as  anti- 
septics, or  attenuating  or  killing  the  micro-organisms, 
as  disinfectants.* 

*  Koch,  "  Ueber  Disinfection."  "  Mittheilungen  aus  dem  k.  Ge- 
sundheitsamte,"  1881,  Bd.  I,  S.  234. 


194  BACTERIOLOGICAL  INVESTIGATION. 

DISINFECTION  WITH  FLUIDS. 

Experiments  concerning  disinfection  with  fluids 
are  made  by  filling  watch-glasses,  crystallization- 
dishes,  or  test-tubes,  with  a  solution  of  the  agent  to  be 
tested  in  different  degrees  of  concentration,  and  by 
introducing  into  some  of  these  threads  of  silk  im- 
pregnated with  bacteria  and  spores,  and  into  others 
threads  containing  bacteria  alone. 

After  different  lengths  of  exposure  the  threads  are 
removed  irom  the  solution  with  a  sterilized  pipette, 
washed  with  sterilized  water,  and  three  or  four 
threads  are  placed  in  a  thick  fluid  nutrient  gelatin, 
which  is  laid  on  a  slide,  or  in  other  tested  nutrient 
media.  For  control,  threads  which  have  not  been 
treated  at  all  are  placed  in  gelatin,  and  others  which 
have  only  been  washed  with  sterilized  water.  The 
difference  in  the  time  of  development,  or  the  com- 
plete non-appearance  of  development,  can  then  be 
directly  observed.  Further  control  is  necessary  by 
introducing  such  silk  threads  under  the  skin  of  ani- 
mals. Carbolic  acid,  e.  g.,  kills  the  anthrax-bacilli 
in  a  1  per  cent  solution  in  two  minutes,  while  it  re- 
quires seven  days  for  a  3  per  cent  solution  to  de- 
stroy their  spores.  The  development  of  the  anthrax- 
spores  is  prevented  by  exposure  to  a  2  per  cent  so 
lution  for  three  days. 

Chamberland  and  Eoux  *  inquired  into  the  attenu- 
ating influence  of  carbolic  acid  by  adding  a  defi- 
nite amount  of  the  agent  to  neutralized  calf-bouillon 
which  was  inoculated  with  anthrax-bacilli.  Carbolic 
acid,  1  to  400,  prevented  development  and  destroyed 
the  bacilli  in  forty-eight  hours  ;  1  to  500  killed  them 
after  five  months ;  1  to  600  only  after  six  months. 
*  "  Comptes  rendus,*'  Bd.  XOVI,  1883,  No.  15. 


GENERAL  BIOLOGICAL  PROBLEMS. 


195 


In  1  to  600  the  bacilli,  after  twelve  days,  killed  only 
guinea-pigs,  rabbits,  and  mice,  and  after  twenty-nine 
days  were  inactive.  The  addition  of  carbolic  acid  in 
the  ratio  1  to  800  prevented  the  formation  of  spores, 
which  first  appeared  when  the  proportion  was  re- 
duced to  1  to  1,200. 

DISINFECTION  WITH   GASES. 

The  following  arrangement  of  the  apparatus  after 
Fischer  and  Proskauer  *  is  recommended  for  labora- 
tory experiments  on  disinfection  with  gases.  A  thick- 
walled  glass  vessel  ( A  A)  of  about  20  litres  capacity 
is  closed  with  a  strong  cover  (ad)  of  vulcanized  rub- 
ber, the  rim  of  which  is  pressed  tight  upon  the  neck 
of  the  vessel  by  means  of  a  narrow  ring.  In  the  cen- 

FIG.  29. 


ter  of  the  cover  is  a  large  perforation,  and  around 
about  this  several  smaller  ones,  which  can  be  closed 

*  "  Ueber  die  Disinfection  mit  Ohlor.  nnd  Brom."    "  Mitthei- 
lungen  aus  dem  k.  Gesundheitsamte,"  Bd.  II,  1884,  S.  228. 


196  BACTERIOLOGICAL  INVESTIGATION. 

simultaneously  by  rubber  stoppers.  The  central  per- 
foration, 5  to  6  cm.  broad,  is  for  the  admission  of  the 
objects  to  be  used  for  the  experiment,  which,  arranged 
so  that  the  gases  may  have  entrance  on  all  sides,  are 
placed  on  sieve-like,  perforated  paraffine  dishes  (nn). 
These  dishes,  whose  diameter  for  easy  introduction  is 
somewhat  less  than  the  central  perforation,  are  fast- 
ened to  a  glass  rod  (pp)  which  passes  through  their 
center  so  that  several  such  dishes  can  be  arranged  one 
above  the  other.  These  are  held  in  place  on  the  glass 
rod  by  means  of  rubber  rings  (rr).  The  glass  rod  it- 
self is  placed  firmly  in  a  rubber  stopper  which  exactly 
closes  the  large  central  opening.  The  glass  tube  (cc\ 
that  reaches  almost  to  the  bottom  of  the  vessel  and  is 
bent  at  a  right  angle,  conducts  into  the  glass  vessel 
the  gas  developed  in  the  apparatus  (C).  The  tempera- 
ture of  the  glass  vessel  is  measured  by  a  thermometer 
(t).  The  glass  tube  (b)  ending  close  under  the  cover 
serves  for  the  removal  of  the  gas  each  time  after  the 
experiment.  If  the  aspirator  (B)  is  set  in  motion, 
any  desired  volume  of  air  can  be  removed.  The  gas 
is  passed  through  an  absorption-fluid  in  B,  in  which 
the  proportion  of  the  volume  of  air  absorbed  to  the 
gas  is  analytically  determined. 

In  the  disinfective  experiments  with  fluids  and 
gases,  dried  silk  threads,  some  free  from  spores,  and 
others  containing  them,  are  freely  exposed  to  the 
agent  in  order  to  observe  the  direct  effects.  In  other 
experiments  the  material  is  packed  in  filter-paper, 
in  potatoes  that  have  been  hollowed  out,  etc.,  in  order 
to  imitate  the  natural  conditions  in  which  access  to 
the  germs  is  more  difficult. 


GENERAL  BIOLOGICAL  PROBLEMS.  197 


DRYING. 

In  order  to  study  the  influence  of  drying,  a  drop 
of  culture-fluid  containing  bacteria  is  placed  upon  a 
cover-glass  and  protected  from  dust,  dried,  and,  after 
an  hour,  a  day,  or  a  week,  a  drop  of  culture-fluid  is 
placed  upon  the  dried  preparation,  and  the  cover- 
glass  with  the  hanging-drop  is  transferred  to  a  moist 
chamber.  The  exhaustion  of  the  nu  trient  fluid  brought 
about  by  the  drying  under  otherwise  favorable  condi- 
tions becomes  perceptible  in  the  anthrax-bacilli  by 
the  development  of  spores  in  the  filaments,  which  after 
months,  on  the  addition  of  nutrient  solutions,  again 
germinate.  In  the  cholera  vibrione,  on  the  contrary, 
no  spores  form,  but,  as  an  indication  of  the  exhaustion 
of  the  nutrient  medium,  a  spirilla-like  thread-forma- 
tion appears.  On  this  account,  the  drying,  after  a 
short  time,  also  produces  death,  so  that  later,  on  the 
addition  of  nutrient  media,  no  development  of  these 
bacteria  occurs. 

The  parasitic  bacteria,  attenuated  directly  or  in 
their  spores,  may  perhaps  afford  protection  against 
the  virulent  bacteria.  If  the  animals  used  for  experi- 
ment have  withstood  the  inoculation  of  the  attenu- 
ated material,  they  are  infected  after  a  longer  or 
shorter  interval  with  virulent  material  in  the  same 
manner. 

THE  ACTION   OF  LOW  TEMPEEATUEE  AND  HIGH  * 
PEESSUEE. 

The  influence  of  low  temperature  and  high  press- 
ure upon  bacteria  can  only  be  studied  with  a  special 
apparatus.  The  previous  statements  as  to  this  are  so 
widely  at  variance  that  I  shall  content  myself  by 
merely  alluding  to  this  subject.  It  is  also  desirable 


198  BACTERIOLOGICAL  INVESTIGATION. 

that  this  phase  of  the  method  should  be  somewhat 
better  studied,  because  of  the  statement  of  the  exist- 
ence of  aerobic  bacteria  at  considerable  depths  in  the 
sea,  and  of  the  resistance  of  the  bacteria  to  a  press- 
ure* of  1,000  atmospheres  artificially  produced. 
Here  also  belong  the  declarations  of  Chauveau  and 
Wossnessenski,  f  that  an  increase  of  pressure  produces 
first  an  attenuating  influence,  and  at  a  certain  degree 
causes  death  in  the  anthrax-bacilli.  While  the  resist- 
ance of  many  bacteria  to  frost  at  a  moderate  freez- 
ing temperature  has  been  certainly  determined,  and 
can  be  easily  proved  by  exposure  of  the  inoculated 
solution  or  the  pure  culture  in  winter,  yet  statements 
vary  considerably  as  to  the  most  extreme  tempera- 
ture below  zero,  C.  Pictet  and  Yung  ^  place  as  the 
lower  limit  108  hours  at  70°  C.,  and  20  hours  at 
130°  C. 

ELECTRICITY. 

According  to  Cohn  and  Mendelsohn,*  electricity  in 
the  form  of  a  physiologically  acting  induction-current 
is  without  influence  upon  bacteria  in  fluids. 

The  action  of  the  constant  current  upon  the  multi- 
plication of  bacteria  in  nutrient  solutions,  and  also 
upon  the  development  of  the  micrococcus  prodigi- 
osus  upon  the  surface  of  potatoes,  depends  upon  the 
strength  of  the  current,  and  was  referred  to  its  elec- 
trolytic action. 

PHOSPHORESCENCE. 

The  phosphorescence  of  bacteria  Ludwig  ||  has  en- 
deavored to  study  by  a  micro-spectral  apparatus. 

*  Certes,  "  Comptes  rendus,"  Bd.  98,  S.  690  und  Y45  ;  Bd.  99,  S. 
885.  t  Ibid.,  Bd.  98,  S.  314.  J  Ibid.,  Bd.  98,  S.  V47. 

*  "Beitrage  zur  Biologie,"  Bd.  Ill,  Heft  1,  S.  141. 

fl  "  Ueber  spectroskopische  Untersuchung  photogener  Pilze." 
"  Zeitschrift  far  wissenschaftl.  Mikroskopie,"  1884,  Bd.  I,  S.  181. 


GENERAL  BIOLOGICAL  PROBLEMS.  199 

LIGHT. 

Engelmann  *  found  that  the  swarm  movements 
of  the  bacterium  photo-metricum  are  dependent  upon 
light,  and  that  the  collection  of  cells  in  the  sun-spec- 
trum is  most  marked  in  ultra-red,  and  decreases  more 
and  more  through  the  green  and  blue  to  the  violet. 

*  "  Arch.  f.  d.  ges.  Physiologie,"  Bd.  30,  S.  95. 


YI. 

SPECIAL  HYGIENIC  INVESTIGATION. 
A.   EAETH. 

IT  was  first  experimentally  determined  by  Schlo- 
sing  and  Muntz  *  that  the  ammonia  formed  in  earth 
from  the  disintegration  of  organic  substances  can  be 
reduced  by  the  action  of  bacteria  to  nitric  acid.  The 
oxidation  of  the  carbon  of  organic  substances  to  car- 
bonic acid  was  likewise  experimentally  proved  by 
Yollny  f  to  be  due  to  micro-organisms.  On  the  con- 
trary, there  occurs  in  layers  of  earth  restricted  from 
the  entrance  of  air  a  reduction  of  the  nitrates,  as  Gu- 
yon  and  Dupetit  J  found,  in  which  sometimes  only  the 
nitrites,  but  at  others  nitrogen,  ammonia,  and  nitrous 
oxide  are  formed  by  the  action  of  bacteria.  These 
experiments  are  performed  by  adding  a  particle  of 
earth  to  a  weak  alkaline  nutrient  solution,  or  to  drain- 
water  which  has  been  sterilized  and  contains  the 
ammoniac  salts,  nitrites  or  nitrates,  while  for  control  a 
particle  is  placed  in  another  glass  which  has  been 
sterilized  by  many  hours'  heating  at  150°  or  160°  C. 
For  bringing  about  the  reduction  of  the  nitrates  the 
arrangement  of  experiments  employed  in  anaerobiosis 
is  used. 

*  "  Comptes  rendus,"  Bd.  LXXVII,  1877,  S.  203  und  353 ;  Bd. 
LXXXIV,  S.  301 ;  Bd.  LXXXIX,  S.  891. 

t  Landw.,  u  Versuchsstationen,"  Bd.  XXV,  1880,  S.  390. 
t  "Comptes  rendus,"  1882,  Bd.  XCV,  S.  644  und  S.  1365. 


SPECIAL  HYGIENIC  INVESTIGATION.          201 

By  this  were  furnished  the  first  positive  grounds 
for  the  biological  explanation  of  the  soil- water  the- 
ory of  disease,  which  had  previously  been  purely  hy- 
pothetical. 

The  direct  determination  of  the  presence  of  patho- 
genic organisms,  or  their  spores,  in  the  earth  was  made 
in  1881  by  Pasteur  and  Koch  *  for  the  bacilli  of  ma- 
lignant oedema.  Nicolaif  cultivated  a  form  of  ba- 
cillus from  the  earth  which  produced  in  mice,  rab- 
bits, and  guinea-pigs  a  fatal  form  of  tetanus,  which 
was  transferable  to  other  animals. 
,  Such  inquiries  make  it  seem  desirable  that  a  more 
exact  study  of  the  bacteria  present  in  the  earth  should 
be  made,  because  in  it  optional  parasitic  bacteria  find, 
with  sufficient  moisture  and  temperature,  the  condi- 
tions necessary  for  existence,  while  the  optional  septic 
bacteria  can  thrive  in  it,  at  least  for  a  time.  The 
obligatory  parasitic  bacteria  can  at  least  be  preserved 
in  the  form  of  permanent  spores  in  the  earth. 

In  order  to  form  the  most  comprehensive  conclu- 
sion possible  as  to  the  presence  in  the  earth  of  bac- 
teria or  germs  of  the  same  capable  of  development, 
the  gelatin-plate  cultures  of  Koch  are  most  advan- 
tageously used,  because  of  reasons  previously  stated, 
in  which  collectively  the  septic  bacteria  and  the  op- 
tional parasites  at  present  known  and  the  optional 
septic  bacteria  can  develop  into  isolated  colonies. 
Since  the  individual  organisms  already  found  in  the 
earth,  as  the  bacilli  of  malignant  oedema  and  of  buty- 
ric-acid fermentation,  are  anaerobic,  the  gelatin-plates 
are. covered  with  sheets  of  mica.  It  is  to  be  ob- 
served also  whether  the  species  develop  from  spores, 

*  "  Mittheilungen  aus  dem  k.  Gesundheitsamte,1'  Bd.  1, 1881,  S.  56. 
f  "  Ueber  infectiosen  Tetanus."   '» Deutsche  med.  Wochenschrift," 
1884,  No.  52. 


202  BACTERIOLOGICAL  INVESTIGATION. 

and  whether  at  a  certain  depth  only  the  bacteria- 
forming  spores,  are  met  with.  The  earth  to  be  used 
in  the  experiment  is  pulverized  in  a  sterilized  mortar, 
with  a  sterilized  pestle,  and  with  a  sterilized  scalpel ; 
some  of  the  fine  powder  is  scattered  upon  the  still 
partially  fluid  gelatin,  which  has  been  poured  oat 
upon  a  plate. 

A  few  fine  particles  of  the  earth  may  also  be  in- 
troduced into  the  liquefied  gelatin  while  still  in  the 
test-tube,  and  thoroughly  mingled  by  shaking,  and 
then  the  gelatin  thus  infected  may  be  poured  upon  a 
plate.  The  control  is  made  by  preparing  plates  in  the 
same  way  from  particles  of  earth  which  have  been 
sterilized  by  two  hours'  heating  at  150°  to  160°  C. 

Strongly  obligatory  parasitic  bacteria  can  be  pres- 
ent in  the  earth  only  in  the  form  of  permanent 
spores,  and  these  can  be  directly  cultivated  from  the 
earth  only  in  a  special  manner.  As  a  rule,  it  is  ne- 
cessary to  use  the  round-about  way  of  the  infection- 
method,  in  order  to  procure  an  original  material  as 
clean  as  possible  for  the  blood-serum  cultures.  Par- 
ticles of  the  earth  are  placed  in  one  or  more  pockets 
beneath  the  skin,  and  then  another  animal  is  infected 
with  the  pus,  blood,  or  tissue-fluid  of  the  sick  animal. 
After  a  few  transfers  the  animals  will  present  in  their 
tissues  pure  cultures  of  the  malignant  form  of  bac- 
teria, and  then  blood-serum  cultures  can  be  prepared 
in  the  previously  described  manner,  as  was  success- 
fully done  by  Mcolai. 

B.  WATEE. 

The  chemical  investigation  of  water  alone  can  fur- 
nish no  information  as  to  the  hygienic  fitness  or  un- 
fitness  of  a  certain  water  for  use  and  drinking.  The 
water  chemically  purest — the  distilled  water  of  the 


SPECIAL  HYGIENIC  INVESTIGATION.  .       203 

laboratory,  for  example — always  contains  bacteria 
and  their  germs ;  and,  on  the  other  hand,  water  toler- 
ably dirty  chemically  may  contain  relatively  few 
micro-organisms.  Chloride  of  sodium,  the  type  of 
the  chemical  substances  from  inhabited  places,  al- 
ways presents  a  valuable  index  of  the  cleanliness  as 
compared  with  good  spring-water,  and  in  the  same 
way  the  nitrites  are  significant,  because  they  afford  in- 
formation (from  the  previously  described  reasons) 
that  the  water  is  in  relation  with  localities  in  which 
the  biological  processes  have  not  yet  come  to  a  rest, 
since  the  nitrites  are  produced  by  oxidation  or  reduc- 
tion. With  freedom  from  its  limitations  and  with 
careful  consideration  as  to  the  local  conditions,  now 
as  formerly,  valuable  conclusions'  may  be  drawn  as 
to  the  hygienic  value  of  the  water  from  the  usual 
chemical  examination. 

But  not  only  the  soluble  matters  of  the  biological 
processes  in  the  earth  may  be  mingled  with  the  water, 
but  also  the  organisms  themselves.  A  priori,  there 
exists  not  the  slightest  ground  for  attributing  to  the 
soil-water  alone,  or  rather  to  the  moistening  of  the 
subsoil  caused  by  the  same,  an  equal  importance  in 
the  existence  and  extension  of  infectious  diseases 
with  the  water  for  use  and  drinking.  The  publication 
of  localized  investigations  has  added  information  to 
both  possibilities. 

The  direct  microscopical  examination  of  water  is 
made  both  in  the  hanging-drop  and  in  the  dried 
cover-glass  preparations.  By  culture-experiments,  on 
the  one  hand,  the  number  of  germs  capable  of  devel- 
opment is  determined,  and  on  the  other  the  presence 
or  absence  of  pathogenic  species  among  them. 

The  water  is  collected  in  sterilized  vessels.  Then 
1  c.  cm.  is  drawn  into  a  sterilized  pipette  and  thor- 


204  BACTERIOLOGICAL  INVESTIGATION. 

oughly  mixed  in  a  test-tube  with  about  10  c.  cm.  of  a 
10  per  cent  nutrient  gelatin  liquefied  at  30  C.  This 
mixture  is  poured  out  upon  a  sterilized  glass  plate, 
which,  after  the  solidification  of  the  gelatin,  is  placed 
in  a  moist  jar. 

In  these  plate-cultures  the  strongly  obligatory 
parasitic  bacteria  can  not  develop,  and  there  are  rare- 
ly so  many  germs  present  that  they  can  not  be  num- 
bered. Notwithstanding  that  the  obligatory  parasitic 
bacteria  do  not  develop  in  plate-cultures,  these  are 
for  hygienic  purposes  of  equal  value,  because  it  is  of 
the  first  importance  in  the  foundation  or  improve- 
ment of  a  water-supply  to  know  whether  the  water 
contains  pathogenic  micro-organisms  which  can  exist 
outside  of  the  body. 

If  the  water  is  very  rich  in  germs,  it  must  be  di- 
luted in  a  definite  manner  with  10,  50,  or  100  to  1,000 
c.  cm.  of  sterilized  distilled  water.  On  practical 
grounds,  in  order  to  determine  a  directly  comparable 
number,  it  is  recommended  by  Koch  *  that  the  num- 
ber of  germs  capable  of  development  in  1  c.  cm.  of 
the  original  water  be  computed. 

By  the  form  of  their  development,  suspected  or 
unknown  species  are  brought  to  a  proof  as  to  their 
malignant  peculiarities  by  inoculation  of  animals. 

If  the  water  can  be  directly  employed  for  ex- 
amination, it  is  best  to  fill  sterilized  flasks,  which 
are  immediately  closed  with  sterilized  cotton  stop- 
pers. For  transportation  the  following  procedure 
is  recommended  :  Small  cylindrical  bottles,  of  about 
100  c.  cm.  capacity,  provided  with  ground-glass  stop- 
pers, are  thoroughly  cleaned  in  the  laboratory  and 

*  Ofr.  auch  Becker's  "  Anleitung  zur  Untersuchung  des  Wassers 
auf  Mikroorganismen."  Borner's  "  Eeichs-Medizinal  Kalendar  fur 
1885,"  Heft  II. 


SPECIAL  HYGIENIC  INVESTIGATION.          205 

sterilized  at  150°  to  160°  C.  Then  a  rubber  cap,  ster- 
ilized by  steam,  is  closely  drawn  over  the  stopper, 
and  the  vessel,  with  the  cap,  is  inclosed  in  a  small 
wooden  box.  For  the  reception  of  water  from  a  sup- 
ply-pipe, after  the  water  has  first  been  allowed  to 
run  for  a  few  minutes,  the  vessel  is  filled  under  the 
faucet,  while  the  rubber  cap  and  the  stopper  are 
held  with  sterilized  fingers.  Then  the  stopper  is  re- 
placed, the  cap  is  drawn  over,  and  the  vessel  is  in- 
closed in  the  box. 

For  obtaining  water  from  a  spring  the  rubber  cap 
is  removed,  and  the  glass  stopper,  withdrawn  under 
water,  is  replaced  again  after  a  minute  or  two ;  the 
cap  is  again  drawn  over  it,  and  the  vessel  is  dried  on 
the  outside  and  placed  in  the  box.  In  this  manner 
any  infection  outside  of  the  water  is  prevented. 

The  number  of  colonies  developing  is  not  always 
equal,  especially  as  every  elevation  of  the  tempera- 
ture is  associated  with  an  increase  in  number  of  the 
germs.  Standing  for  one  day  in  a  warm  room  has  an 
influence  in  this  way.  In  the  same  way  as  the  num- 
ber of  germs  which  develop  varies,  the  number  in 
the  different  forms  also  varies  considerably.  The 
judgment  in  the  bacteriological  side  of  water  investi- 
gation presupposes  previous  studies  in  bacteriology. 

c.  AIE. 

In  order  to  determine  the  germs  present  in  the 
air,  Pasteur  caused  the  air  to  pass  through  gun-cot- 
ton ;  then  dissolved  the "  gun-cotton  in  ether,  and 
examined  the  solution  microscopically.  Miquel  *  en- 
deavored, with  improvements  of  the  previous  appa- 
ratus, to  number  the  micro-organisms  directly.  In  a 
jar,  with  the  opening  looking  downward,  a  hollow 
*  "  Annuaire  de  1'observatoire  de  Montsouris." 


206  BACTERIOLOGICAL  INVESTIGATION. 

ball  is  screwed,  which  has  a  fine  opening  at  the  apex. 
Opposite  to  this  opening  in  the  jar  a  cover-glass  is 
placed,  which  is  smeared  with  glycerine  and  sugar. 
If  the  air  is  aspirated  into  the  jar,  it  must  first  strike 
upon  the  cover-glass  and  surrender  its  dust  to  the 
sticky  fluid,  which  is  then  microscopically  examined. 
For  numbering  the  bacteria,  Miquel  causes  a  measured 
volume  of  air  to  pass  through  a  sterilized,  neutralized 
solution  of  beef -extract. 

Emmerich  *  devised  an  improvement  of  the  aero- 
scope  for  fluids.  A  pear-formed  glass  (Fig.  30)  com- 
municates by  a  fine  opening  with  a  glass 
tube,  70  or  80  cm.  long,  which  leads  to 
a  glass  ball  by  about  seven  gently  ris- 
ing, parallel  spirals.  The  apparatus  is 
filled  with  20  or  25  c.  cm.  of  a  nutrient 
solution,  and  the  air  is  drawn  through 
by  aspiration,  which,  in  small  bubbles 
from  the  opening  for  entrance,  passes 
out  by  the  long  way  through  the  spiral, 
so  that  it  comes  in  intimate  contact 
with  the  solution,  and,  on  this  account, 
can  easily  surrender  its  germs  to  it. 

Since,  also,  in  the  complete  absorption  of  the 
germs  by  the  sterilized  nutrient  solution,  serving  as 
a  wash-fluid,  an  isolated  development  of  the  germs, 
and  thus  a  direct  enumeration,  is  impossible,  the 
practicability  of  all  aeroscopes  with  the  use  of  fluid 
ceases  where  really  a  beginning  should  be  made. 
The  possibility  of  testing  a  fluid  laden  with  germs 
as  to  its  contents  in  germs  is  rendered  practicable 
by  mingling  these  solutions  with  sterilized  nutrient 
gelatin,  so  that  then  the  individual  germs  isolated 
can  develop  into  colonies  in  the  gelatin. 

*  "  Archiv  fur  Hygiene,"  Bd.  I,  1883,  S.  169. 


SPECIAL  HYGIENIC  INVESTIGATION. 


207 


FIG.  81. 


After  Koch  *  had  demonstrated  in  principle  the 
value  of  nutrient  gelatin  for  the  examination  of  air 
also,  Hesse  f  constructed  the  following  apparatus, 
which  consists  of  a  long  glass  tube  and  an  aspirator. 
The  glass  tube  (B,  Fig.  31)  has  a  length  of  about  70 
cm.  and  a  diameter  of  3^ 
cm.  The  end  (a)  is  closed 
by  a  closely  fitting  rubber 
cap,  provided  with  a  cen- 
tral round  perforation, 
and  over  this  a  second 
similar  cap  is  placed, 
which  has  no  perforation, 
and,  on  this  account,  com- 
pletely closes  the  end  of 
the  tube.  The  end  (5)  is 
provided  with  a  tightly 
fitting  rubber  stopper,  in 
the  center  of  which  is  a 
hole  about  1  cm.  wide. 
Through  this  a  glass  tube, 
about  10  cm.  long,  is  in- 
troduced, the  free  end  of  which  is  connected  with  the 
aspirator  (A  and  A').  In  this  glass  tube  two  cotton 
stoppers  are  placed,  one  of  which  lies  near  the  center, 
while  the  other  projects  inward  somewhat  into  the 
lumen  of  the  tube. 

After  removal  of  the  rubber  stopper  the  tube  is 
filled  with  about  50  cm.  of  a  sterilized  nutrient  gela- 
tin (containing  5  to  10  per  cent  of  gelatin),  the  end  is 
closed  again,  and  the  tube  with  the  gelatin  placed 

*  "  Mittheilungen  aus  dem  kaiserlichen  Gesundheitsamte,"  Bd.  I, 
1881,  S.  32. 

f  "  Ueber  quantitative  Bestimmung  der  in  der  Luft  enthaltenen 
Mikroorganismen."     "  Mittheilungen,"  Bd.  II,  1884,  S.  182. 
14 


208  BACTERIOLOGICAL  INVESTIGATION. 

for  one  to  two  hours  in  hot  steam.  After  the  tube 
has  cooled  enough,  so  that  it  can  be  handled  readily, 
the  entire  inner  surface  of  the  tube  to  B  is  coated 
with  a  thin  layer  of  gelatin,  while  the  bottom,  upon 
which  the  germs  will  almost  exclusively  settle,  is 
clothed  with  a  greater  mass  of  gelatin.  For  this 
purpose  the  tube  is  cooled  under  the  water-faucet 
by  successively  bringing  it  in  its  entire  length  hori- 
zontally under  the  stream,  and  then  rotating  it  rap- 
idly upon  its  axis.  When  the  gelatin  becomes  quite 
consistent  the  rotation  is  immediately  ceased,  and 
it  is  then  moved  only  horizontally.  Thus  on  one 
side,  in  its  entire  length,  a  somewhat  thicker  layer 
of  gelatin  is  formed,  which,  in  putting  the  tube  on  its 
support,  is  placed  downward  as  the  floor  layer. 

At  the  place  for  the  observation  the  outer  cap 
is  removed  from  the  end  («),  the  aspirator  is  set  in 
motion,  and  the  air  is  slowly  drawn  through.  In  the 
experiments  in  the  open  air  two  separated  litre-flasks 
can  be  used  as  an  aspirator. 

In  order,  on  the  one  hand,  to  meet  the  objection 
that  not  all  the  germs  cling  to  the  surface  of  the  gela- 
tin, but  especially  for  the  improvisation  of  an  appa- 
ratus which  unites  the  advantages  of  the  gelatinizing 
substances  with  a  wash-fluid,  recourse  may  be  had  to 
the  following  procedure :  Sterilized  test- tubes  are  pro- 
vided with  rubber  stoppers  that  have  been  sterilized 
in  steam,  and  in  which  are  two  perforations.  In  both 
of  these,  sterilized  glass  tubes,  bent  at  right  angles,  are 
introduced  ;  one  of  the  tubes  ends  directly  under- 
neath the  stopper,  while  the  other  passes  almost  to 
the  bottom.  The  test-tubes,  thus  armed,  are  filled  with 
nutrient  gelatin,  or,  according  to  von  Sehlen,*  with 
agar-agar  which  has  been  carefully  sterilized.  The 

*  "  Studien  fiber  Malaria."   "  Fortschritte  der  Medizin,"  1884,  No.  18. 


SPECIAL  HYGIENIC  INVESTIGATION.          209 

free  ends  of  the  glass  tubes  are  closed  by  sterilized 
cotton  stoppers,  which  are  removed  at  the  spot  from 
which  the  air  is  to  be  taken.  The  gelatin  is  liquefied 
by  placing  it  in  water  at  30°  C.,  the  agar-agar  at  40°  C., 
and  during  the  aspiration  it  is  held  in  solution  at  this 
temperature,  so  that,  as  a  nutrient  solution,  it  re- 
ceives the  germs. 

Care  must  be  taken  to  join  two  or  three  such  test- 
tubes  with  one  another,  since  if  a  large  amount  of  air 
is  drawn  in,  the  first  tube  will  not  contain  all  the 
germs  of  the  air.  After  completion  of  the  experi- 
ment the  cotton  stoppers  are  replaced  and  the  solu- 
tions solidified  by  removing  them  from  the  warm 
bath.  In  order  to  prevent,  in  these  cases,  any  fur- 
ther infection,  the  germs  are  allowed  to  develop  iso- 
lated in  the  test-tubes. 


VII. 

BACTERIOLOGY  AS  AN  OBJECT  OF  INSTRUCTION. 

IF  the  methods  thus  far  developed  for  the  inves- 
tigation of  bacteria  should  obtain  the  significance 
which  the  mass  of  physicians  ascribe  to  these  things, 
then  our  high-schools  must  furnish  the  opportunity 
for  instruction  in  this  important  subject. 

Up  to  this  time  this  has  been  done  in  a  quite  in- 
sufficient manner,  from  the  lack  of  suitable  instructors 
and  institutes.  Great  praise  is  due  the  University  of 
Copenhagen  for  going  in  advance,  breaking  the  way, 
and  for  having  established  in  1883,  in  connection  with 
the  Botanical  Institute,  an  institute  for  medical  bac- 
teriology, the  direction  of  which  was  discreetly  placed 
in  the  experienced  hands  of  Salomonsen.  Of  the 
German  universities,  as  yet  only  Munich  in  connec- 
tion with  the  pathological  institute,  Gottingen  in  the 
hygienic,  and  Breslau  in  the  botanical  institute,  fur- 
nish the  opportunity  for  learning  the  methods.  In 
addition  to  these  there  is  still  the  hygienic  bacterio- 
logical department  of  the  chemical  institute  of  Frese- 
nius  at  Wiesbaden,  directed  by  myself,  which  takes 
teaching  into  special  consideration.  The  laboratory 
of  the  office  of  the  Imperial  Board  of  Health  is  only 
accessible  in  a  civil  and  diplomatic  way,  so  that  for 
the  general  purpose  of  teaching  it  does  not  come  at 
all  into  consideration. 


BACTERIOLOGY  AS  AN  OBJECT  OF  INSTRUCTION.  211 

The  zeal  with,  which  the  work  on  this  subject  is 
now  done  at  all  high-schools  would  be  exceedingly 
fruitful,  and  would  render  many  a  fiasco  impossible, 
if  an  opportunity  for  work  in  the  methods  were  af- 
forded in  a  manner  sufficient  for  the  existing  needs. 
The  demand,  as  such  institutes  are  constituted,  is  at 
the  moment  scarcely  to  be  answered  in  a  sufficient 
manner,  from  the  want  of  suitable  instruction  and 
from  pecuniary  considerations. 

Botany  is  interested  in  the  solution  of  many  mor- 
phological and  biological  questions  in  bacteriology. 
(But  a  distinguished  botanist  has  sorrowfully  com- 
plained of  the  disgrace  that  most  botanists  do  not 
concern  themselves  with  bacteriology.)  These  ques- 
tions can  be  referred  to  the  botanical  institutes,  of 
which  there  is  no  lack. 

It  is  impossible  for  physiology  to  be  satisfied  with 
the  present  condition  of  the  subject  of  digestion,  but 
it  must  now  give  quite  a  different  consideration  than 
formerly  to  the  biological  processes  in  the  intestines. 
The  solution  of  these  problems,  which  are  associated 
in  part  with  the  physiology  of  fermentation,  can  be 
referred  exclusively  to  the  richly  endowed  physiologi- 
cal and  physiologico-chemical  institutes. 

Also  pathology,  sufficiently  provided  with  insti- 
tutes among  the  many  attractions  with  which  it  has 
to  do,  can  not  fail  to  give  greater  consideration  to 
bacteria  as  a  further  field  for  investigation. 

Whoever  has  really  interested  himself  in  bacteria 
investigation  is  rejoiced  when  the  subject  is  under- 
taken from  the  most  different  standpoints.  But  such 
divergent  standpoints  do  not  suffice  for  the  investi- 
gation, if  they  do  not  succeed  in  giving  to  bacteri- 
ology its  due  place  as  an  object  of  study. 

For  a  definite  basis  for  bacteriology  it  seems  to  me 


212  BACTERIOLOGICAL  INVESTIGATION. 

that  only  two  ways  offer  a  prospect  for  lasting  re- 
sults :  first,  special  bacteriological  institutes  can  be 
founded,  as  at  Copenhagen  ;  or,  secondly,  these  insti- 
tutes ought  to  be  united  with  the  hygienic  institutes 
to  be  erected.  The  latter  seems  to  me  to  be  the  most 
promising  plan. 

Botany,  physiology,  and  pathology  have  always 
an  actual  interest  only  on  a  certain  side  of  the  inves- 
tigation, while  to  the  other  questions  they  are  placed 
in  nearly  equal  relation. 

The  botanical  questions  possess  an  important  in- 
terest for  hygiene,  because  the  formation  in  the  sys- 
tem, the  constancy  and  the  variability  of  forms,  etc., 
can  have  a  direct  influence  upon  the  hygienic  treat- 
ment. Hygiene  shares  this  interest  in  botanical 
questions  in  part  with  physiology  and  pathology. 
Hygiene  must  know  the  general  biology  of  bacte- 
ria, because  the  general  processes  of  decomposition 
brought  about  by  septic  micro-organisms  are  of  sig- 
nificance in  the  consideration  of  the  aetiology  of  dis- 
ease. Botany  and  physiology  in  part  share  in  this 
interest. 

Hygiene  must  be  very  familiar  with  the  patho- 
genic bacteria,  since  these  have  not  only  the  value 
perhaps  of  an  external  attraction  for  the  aetiology, 
but  because,  as  the  conditio  sine  qua  non,  they  form 
the  hinge  of  the  entire  aetiology,  which  pathology  can 
consider  only  in  an  insufficient  manner,  because  the 
processes  in  the  body  lay  claim  sufficiently  to  their 
activity. 

The  problems  of  combining  the  experimental  inves- 
tigations concerning  the  parasites  of  disease  with  the 
results  of  the  investigations  as  to  the  accessory  causes 
of  epidemic  diseases  (basing  the  hypothesis  of  the 
soil  and  drinking-water  theories  upon  the  biology  of 


BACTERIOLOGY  AS  AN  OBJECT  OF  INSTRUCTION.  213 

micro-organisms  and  facts,  and  no  longer  exclusively 
upon  reflections  and  probabilities)  belong  only  to 
hygiene,  and  are  to  be  solved  in  a  satisfactory  man- 
ner only  by  looking  well  to  all  sides  of  the  investiga- 
tion, few  parts  of  which  are,  after  all,  only  sufficiently 
recognized  by  botany,  physiology,  and  pathology. 
It  seems  to  me  to  follow,  from  these  considerations, 
that  that  branch  of  science  which  from  its  very  na- 
ture must  be  accountable  to  the  whole  field  is  best 
adapted  in  the  fullest  sense  to  do  justice  to  the  sub- 
ject in  hand  as  an  object  of  teaching. 

In  the  same  manner  as  the  teacher  of  hygiene,  in 
another  direction,  is  required  to  familiarize  himself 
with  bacteriology  to  enable  him  to  impart  the  same 
to  his  pupils,  so  must  we  also  require  of  the  bacteri- 
ologist intrusted  with  hygiene  that  he  should  master 
the  other  chapters  of  hygiene  to  become  a  teacher. 

Hygiene  can  not  and  must  not  be  lost  sight  of  in 
the  importance  of  bacteriology,  and  it  would  be  a  sad 
error  to  consider  only  the  bacteriological  side  in  fill- 
ing the  chair  of  hygiene.  Bacteriology  as  an  object 
of  study,  as  seen  in  some  institutions,  would  be  more 
practical  if  combined  with  hygiene. 

It  is  self-evident  that,  in  the  methods  of  teaching, 
two  things  must  be  kept  well  separated — i.  e.,  the  in- 
troduction into  the  methods  by  practical  courses,  and 
independent  working  after  preparatory  instruction ; 
the  latter  will  only  be  employed  by  a  few  especially 
interested,  but  it  must  be  sufficiently  observed  in  the 
equipment  of  the  institution.  The  actual  every-day 
necessities  lie  at  present  more  in  the  preparatory 
courses.  Every  practicing  physician,  and  especially 
every  medical  officer,  is  desirous  of  so  far  informing 
himself  concerning  these  methods  as  to  be  able  to  fol- 
low intelligently  the  recent  investigations,  since  the 


214:          BACTERIOLOGICAL  INVESTIGATION. 

morphological  and  biological  foundations  of  the  gen- 
eral processes  of  decomposition  and  the  aetiology  of 
infectious  diseases  have  assumed  tangible  form 
through  the  development  of  the  methods. 

Courses  may  be  arranged  in  the  form  of  practical 
exercises  lasting  from  four  to  six  weeks,  in  which 
the  most  important  methods  are  practiced  with  par- 
ticular regard  to  those  most  important,  considered 
from  a  hygienic  standpoint.  This  plan  alone  might 
at  the  time  be  adequate  to  meet  the  necessities  of 
medical  officers  and  any  surgeons  who  can  absent 
themselves  from  duty  for  only  a  few  weeks,  but  is 
far  less  agreeable  to  the  teacher  than  if  he  could  dis- 
tribute the  practical  introduction  into  the  methods 
over  a  semester.  The  latter  plan,  which  must  be- 
come the  rule  for  the  young  medical  man  and  the 
naturalist,  permits  the  teacher  to  take  cognizance  of 
all  methods,  and  thus  to  render  the  perception  more 
acute  for  the  development  and  for  the  future  prob- 
lems of  investigations. 

As  a  result,  this  mode  of  introduction  into  the 
methods  becomes  an  important  aid,  which  well  pre- 
pares for  independent  work,  and  may  bring  about 
the  entire  disappearance  of  over-hasty  publications 
in  bacteriology. 


INDEX. 


ABBE,  condenser  of,  41. 

immersion-fluid  of,  35. 
Acetate  of  potash  for  preservation, 

53. 
Acetic  acid  for  maximal  decoloriza- 

tion,  52. 

Acid  aniline-dyes,  46. 
Acromatic  condenser,  35. 
Actinimycosis,  90. 
Aerobic  bacteria,  10. 
Agar-agar  for  cultures,  144. 
Air,  bacteria  in  the,  205. 
Anaerobiosis  in  fluids,  164. 
Aniline-dyes  for  staining  bacteria,  43. 
Aniline-water  solution,  49. 
Anise-oil  for  immersion  system,  36. 
Anthrax  bacteria,  attenuation  of,  190. 

effect  of  drying,  197. 

accidentally  parasitic,  158. 
ARSONVAL,  D',  thermostat  of,  188. 
Arthro-bacteria,  29. 
Arthro-cocci,  29. 
Arthrospore  bacteria,  10. 

Bacilli,  29. 

Bacteria,  forms  of,  28. 

Basic  aniline-dyes,  46. 

BAUMGARTEN,  leprosy  bacillus,  65. 

Biological  problems,  general,  183. 

Blood,  examination  for  bacteria,  67. 

inoculation  from,  infra  vitam,  155. 
Blood-serum  for  cultures,  148. 
Brood-oven  for  high  temperatures,  99. 
BREFELD,  gelatin  to  prevent  evapora- 
tion of  culture-fluids,  129. 

studies  from  a  single  germ,  107. 

transparent  fluid  media  of,  128. 


Canada  balsam  for  mounting,  63. 

Capillary -tubes  for  cultures,  121. 

Capsule  cocci  of  pneumonia,  staining 
of,  86. 

Caraghen  for  cultures,  144. 

Carbonate  of  soda  to  neutralize  nu- 
trient gelatin,  132. 

Carmine     for     staining    micrococci, 
42. 

CARTER,  infection  of  monkeys  with 
relapsing  fever  spirillum,  126. 

Causal  relation  of  bacteria  to  disease, 
160. 

Cedar  oil  for  immersion  system,  36. 

Cement,  vermilion  paint,  24. 

Chamber,  moist,  for  pure  cultures, 
135. 

Chicken  cholera  bacillus,  attenuation 
of,  190. 

Cholera  Asiatica  bacilli,  effect  of  dry- 
ing, 197. 
staining  in  sections,  88. 

Chromic-acid  solution,  52. 

Cladothrix,  30. 

Clostridium,  29. 

Cloves,  oil  of,  53. 

Cocci,  29. 

COHN,  artificial  culture-fluid  of,  95. 
transparent  fluid  media  of,  1 28. 

Colony  from  a  single  germ,  131. 

Colonies,  isolated,  130. 

Comma-bacilli,  32. 

Condenser  of  ABBE,  43. 

Copenhagen,  University  of,  210. 

Counting-slide  of  THOMA,  116. 

Cover-glass  preparations,  55. 

Culture-methods,  92. 


216 


INDEX. 


Culture-oven  for  high  temperatures, 

99. 
Cutaneous  inoculation,  177. 

Decalcify  ing  solutions,  52. 
Desmo-bacteria,  29. 
Dilution,  method  of,  113. 

for  plate  cultures,  141. 
Discontinuous  sterilization,  20. 
Disinfection  with  gases,  197. 

with  fluids,  195. 
Distilled  water,  use  of,  53. 
Drying,  influence  of,  on  bacteria,  197. 

Earth,  bacteria  in,  200. 
EHRLICH-WEIGERT-KOCH    method    of 

staining  tubercle  bacilli,  65. 
Electricity,  effect  on  bacteria,  198. 
Endospore  bacteria,  29. 
Enzyme,  185. 
Eosin  for  staining,  46. 
Epiphytic  bacteria,  88. 
Erysipelatous  skin,  inoculation  from, 

156. 
Eye,  inoculation  of  anterior  chamber 

of,  179. 

Fat  crystals  are  pseudo-bacteria,  64. 
Feeding  experiments,  75. 
Ferro-cyanide,  double,  169. 
Filter  warmed  with  hot  water,  133. 
FITZ,   development  from  one  germ, 

128. 

Flagella,  staining  of,  73. 
Fluid  culture-media,  92. 
Fluorescin  for  staining  bacteria,  46. 
Fractional  cultures,  100. 
FRIEDLANDER'S  pneumococci,  staining 

of,  86. 
Fuchsin  for  staining  bacteria,  46. 

GAY-LUSSAC,  experiments  on  spon- 
taneous generation,  15. 

GEISSLER'S  culture-chamber,  105. 

Gelatin  for  solid  transparent  culture- 
media,  preparation  of,  130. 

General  biological  problems,  183. 

Gentian-violet  for  staining  bacteria, 
46. 

Glanders,  bacilli  of,  staining,  86. 

Gonidia,  29. 

GRAM'S  method  of  decolorization, 
61. 

Granular  detritus  vs.  bacteria,  88. 


Haematoxylin  for  staining,  42. 
Hanging-drop,  39. 
Heat,  method  of  isolation  by,  119. 
HESSE,  apparatus  of,  for  examination 

of  air,  207. 

Homogeneous  immersion-lens,  37. 
Hydrobiosis,  163. 
Hygienic  investigation,  special,  200. 

KLEBS,  use  of  gelatin  to  prevent  evap- 
oration of  culture-fluids,  129. 

method  of  inoculation,  100. 
KOCH,  attenuation  of  anthrax  bacilli, 
191. 

blood-serum  for  cultures,  148. 

the  color-  vs.  the  structure-picture, 
41. 

pure  culture  of  tubercle  bacilli,  128. 

examination  for  tubercle  bacilli,  61. 

homogeneous  immersion  system,  36. 

infection  of  monkeys  with  spirilla 
of  relapsing  fever,  126. 

modification  of  PRAVAZ  hypodermic 
syringe,  179. 

steam  sterilization-cylinder  of,  18. 

Lactic-acid  bacteria,  190. 

Leprosy  bacilli  like  tubercle  bacilli, 

65. 

staining  of,  in  sections,  87. 
Leptothrix,  29. 
Light,  effect  on  bacteria,  199. 
Liquefaction  of  gelatin  by  bacteria, 

137. 
LISTER,  development  from  one  germ, 

128. 
LOFFLER,  meat-water  peptone  gelatin, 

133. 

Malignant  redema,  bacilli  of,  201. 
Meat-water  peptone  gelatin,  132. 
Methyl-blue  for  staining  bacteria,  46. 
Methyl-violet  for  staining  bacteria,  46. 
Mica,  use  for  cultures  of  anaerobic 

bacteria,  163. 
Mice,  field-  and  house-,  septicaemia  of, 

126. 

Microscopical  technique,  28. 
MIQUEL,  examination  of  the  air  for 

bacteria.  205. 
Moist    chamber    for    pure    cultures, 

135. 

Mould-fungi,  cultivation  of,  158. 
Movements  of  bacteria,  38. 


INDEX. 


217 


MULLER'S  fluid,  62. 
Mycosis,  90. 

NAEGELI,  artificial  culture-fluid,  96. 
development  from  one  germ,  128. 
Nail-cultures  of  FRIEDLANDER,  143. 
Nutrient  gelatin,  132. 

Obligatory  anaerobic  bacteria,  10. 
Optional  anaerobic  bacteria,  10. 
Oil  of  cloves,  cedar,  etc.,  53. 
Opaque  solid  culture-media,  101. 
Origin  from  one  germ,  105. 

Parasitic  bacteria,  9,  170. 

PASTEUR,  attenuation  of  bacteria  of 
anthrax  and  chicken  cholera,  190. 
artificial  culture-fluid  of,  94. 
examination  of  the  air  for  bacteria, 

205. 
experiments    on    anaerobiosis    in 

fluids,  166. 

spontaneous     generation     experi- 
ments of,  17. 

Pathology  of  plants,  bacteria  in,  181. 

Peptone  added  to  nutrient  gelatin, 
133. 

Phosphorescence,  effect  of,  on  bacte- 
ria, 198. 

Picric  acid  for  staining,  46. 

Picro-carmine  for  staining,  43. 

Plants,  pathology  of,  181. 

Plate-cultures,  138. 

Pneumococci,  staining  of,  86. 

Potassium,  acetate  of,  for  preserva- 
tion, 53. 

Potato-cultures,  102. 

PRAVAZ  subcutaneous  syringe,  179. 

Pressure,  high,  effect  of,  on  bacteria, 
197. 

Pseudo-bacilli,  64. 

Ptomaines,  187. 

Pyrogallic  solution  for  experiments  in 
anaerobiosis,  166. 

Quantity-culture,  101,  105,  118,  121, 
157,  158,  165. 

Rabbit  septicaemia,  126. 
RECKLINGHAUSEN,  VON,  culture-cham- 
ber of,  105. 

Relapsing  fever,  spirillum  of,  stain- 
ing, 88. 
spirillum  of,  in  monkeys,  126. 


Safranin  for  staining,  46,  86. 
SALOMONSEN,  at  Copenhagen,  210. 
capillary-tubes    of,    for    cultures. 

121. 
isolation  of  pure  colonies  in  blood, 

123. 

Saprophytic  bacteria,  9,  11. 
SCHWANN,  spontaneous  generation  ex- 
periments of,  16. 
Sections,  preparation  of,  78. 
Septic  bacteria,  9,  160. 
Septicaemia  of  mice,  126. 

of  rabbits,  126. 

Serum,  blood-,  for  cultures,  148. 
Slide-cultures,  134. 
Solid  culture-media,  opaque,  101. 

transparent,  128. 
SPALLANZANI,  spontaneous  generation 

experiments  of,  15. 
Spirilla,  29. 
Spirochaetaa,  29. 

Spontaneous  generation,  15,  25. 
Spores,  relation  of,  to  temperature, 

191. 

method  of  staining,  74. 
Sputum    examination    for    tubercle 

bacilli,  61. 

Staining  bacteria,  methods  of,  40. 
Sterilization,  by  discontinuous  heat- 
ing, 20. 

steam-cylinder  for,  18. 
steam-kettle  for,  17. 
principles  of,  15. 
Sterilizing  oven,  26. 
"  Structure-picture,"  34. 
Subcutaneous  applications,  178. 
injections,  179. 
syringe  of  PRAVAZ,  179. 

Technique,  microscopical,  28. 
Temperature,  behavior  of  bacteria  to, 
188. 

effect  of  low,  197. 

relation  of  spores  to,  191. 
Test-tube  cultures,  142. 
Thermostat  of  D'ARSONVAL,  188. 
THOMA,  counting-slide  of,  116. 
TIEGHEM,  VAN,  infection  of    plants, 

125. 

Torula,  29. 
Transparent  fluid  culture-media,  92. 

solid  culture-media,  129. 
Triangle  to  level  plate-cultures,  135. 
Trichinosis,  125. 


218 


INDEX. 


Tubercle    bacilli    demonstrated    by 

caustic  potash,  38. 
examination  of  sputum  for,  61. 
in  sections,  87. 
Tuberculous     material,     inoculation 

from,  154. 
TYNDALL,    method  of    discontinuous 

sterilization  of,  20. 
Typhoid  bacilli,  staining  of,  86. 

Unstained  bacteria,  examination  of, 
34. 


Vermilion-paint  cement,  24. 
Vesuvin  for  staining,  46. 
Vibriones,  29. 

Water,  bacteria  in,  202. 

WBIGERT,  methods  of  staining  of,  42. 

Yeast,  cultivation  of,  158. 

ZAHN,  pipette  of,  for  blood-cultures, 

124. 

ZEISS,  immersion-lenses,  36. 
Zooglea,  29. 


THE  END. 


THE  DISEASES  OE  THE 
STOMACH. 

BY  DR.   C.  A.   EWALD, 

EXTRAORDINARY  PROFESSOR   OF  MEDICINE  AT  THE  UNIVERSITY  OF  BERLIN; 
DIRECTOR   OF   THE   AUGUSTA   HOSPITAL,   ETC. 


AUTHORIZED  TRANSLATION 

FROM  THE  SECOND  GERMAN  EDITION,  WITH  SPECIAL  ADDITIONS 
BY  THE  AUTHOR, 

BY  MOEEIS  MA1STGES,  A.M.,  M.D., 

ATTENDING  PHYSICIAN  TO  OUTDOOR  DEPARTMENT,  MOUNT  SINAI  HOSPITAL, 
NEW  YORK  CITY,  ETC. 


WITH    THIRTY    ILLUSTRATIONS. 

8vo,  497  pages.     Cloth,  $5.00  ;   Sheep,  $6.00. 


SOLD   ONLY  BY  SUBSCRIPTION. 


The  following  are  extracts  from  a  few  of  the  notices  that  have  appeared  in  the 
medical  press : 

"...  We  can  recall  no  work  on  this  special  subject  published  in  late  years  which  is 
so  thoroughly  well  written  and  useful  as  this  of  Ewald's.  .  .  .  Recent  therapeutics  of  dis- 
eases of  the  stomach  are  discussed  with  candor  and  justice,  yet  critically.  The  author  is  an 
authority  on  what  he  writes.  We  believe  him  to  be  the  best  authority.  .  .  .  Some  of  his 
suggestions  we  have  utilized  and  find  them  excellent." — Texas  Courier- Record  of  Medicine. 

"  The  profession  are  deeply  indebted  to  Dr.  Manges  for  this  excellent  translation  of  Dr. 
Ewald's  lectures  on  diseases  of  the  stomach,  enriched  by  notes  of  the  translator,  revised  by 
the  author,  which  renders  the  work  still  more  acceptable  to  the  American  reader.  .  .  .  The 
various  diseases  of  the  stomach  are  discussed  with  the  detail  and  scientific  accuracy  for  which 
the  author  is  noted,  and  the  work  forms  a  very  valuable  addition  to  the  library." — New  York 
Medical  Times. 

".  .  .  There  is  no  question  but  that  Dr.  Ewald  has  given  us  a  work  of  great  scientific  value, 
and  uf>  to  date  in  every  particular.  This  is  a  book  quite  as  valuable  _to  the  surgeon  as  to  the 
physician,  and  no  progressive  medical  man  can  afford  to  be  without  it." — Omaha  Clinic. 

" .  .  .  The  whole  of  this  very  important  and  difficult  field  is  covered  in  a  way  that  can 
not  fail  to  commend  itself  to  all  physicians  who  desire  to  enlarge  their  sphere  of  professional 
knowledge  and  utility." — Denver  Medical  Times. 

u  The  purchaser  is  impressed  by  the  elegance  of  publication  on  opening  this  book,  and 
such  excellence  is  well  fitted  to  such  a  work  as  this  one  is.  ...  The  work  is  thoroughly 
practical,  and  the  physician  who  does  not  secure  and  carefully  read  it  will  deprive  himself 
of  a  most  important  help  in  the  treatment  of  diseases  of  the  stomach."—  Virginia  Medical 
Monthly.  . ( 

New  York :  D.  APPLETON  &  CO.,  72  Fifth  Avenue. 


THE  ANATOMY  AND 

SURGICAL  TREATMENT 

OF  HERNIA. 

BY  HEKRY  O.  MAKCY,  A.  M.,  M.  D.,  LL.  D., 

LATE  PRESIDENT  OF  THE  AMERICAN  MEDICAL  ASSOCIATION,  ETC. 


ILLUSTRATED 

With  Seventy  full-page  Heliotype  and  Lithographic  Reproductions  from  Cooper,  Scarpa, 

Cloquet,  Camper,  Darrach,  Langeribeck,  Cruveilhier,  and 

others  of  the  Old  Masters, 

AND    THIRTY-FOUR    WOODCUTS   IN   THE   TEXT. 


SOLD  ONLY  BY  SUBSCRIPTION.     HALF  MOEOCCO,  $15.00. 


rpHE  author  has  reviewed,  in  extenso,  the  normal  anatomy  of  the  parts  involved 
-••  in  Hernia,  and  the  remote  causes  which  tend  to  produce  it.  The  pathological 
changes  incident  to  the  more  marked  condition  are  clearly  defined,  and  the  chap- 
ters devoted  to  the  discussion  of  these  subjects  are  very  copiously  illustrated.  In- 
strumental supports  are  carefully  discussed,  and  their  better  methods  of  application 
defined.  All  the  various  methods  of  modern  operation  are  given  in  detail,  and,  as 
far  as  possible,  a  compilation  of  the  results  obtained  under  modern  antiseptic  pro- 
cesses is  made.  The  chapter  devoted  to  the  Animal  Suture  is  worthy  of  especial 
consideration,  since  it  clearly  details  one  of  the  greatest  innovations  of  modern 
surgery  of  universal  value. 

It  is  estimated  that  there  are  between  three  and  four  millions  of  people  in  the 
United  States  alone  suffering  from  Hernia.  Hundreds  of  thousands  of  trusses  are 
manufactured  annually.  Every  physician  is  aware  that  a  hernia  is  a  gradually  in- 
creasing disability,  and  that  it  is  very  rarely  cured  except  by  operative  measures. 
Serious  complications  and  dangers  are  ever  present  to  the  individual  suffering  from 
Hernia,  and  statistical  tables  show  that  the  resulting  mortality  is  very  large.  No 
other  surgical  disability  is  so  liable  to  come  under  the  notice  of  the  physician  as 
Hernia,  and  the  author  holds  that  it  is  in  the  highest  degree  the  duty  of  every  prac- 
titioner to  familiarize  himself  thoroughly  with  the  subject.  The  opinion  that  pro- 
fessional obligations  are  discharged  when  the  patient  suffering  from  Hernia  is 
relegated  to  the  instrument-maker  is  erroneous.  The  belief,  as  taught  by  authors 
of  the  last  generation,  that  operative  measures  should  not  be  taken  except  as  a  last 
resort,  because  of  the  attendant  dangers,  has  been  controverted  by  the  achievements 
of  modern  surgery,  among  which  none  are  more  noteworthy  than  the  perfected 
operations  for  the  cure  of  Hernia. 


New  York :  D.  APPLETON  &  CO.,  72  Fifth  Avenue. 


A  TEXT-BOOK  OF 

ANIMAL   PHYSIOLOGY, 

WITH  INTRODUCTORY  CHAPTERS  ON  GENERAL  BIOLOGY,  AND 
A  FULL  TREATMENT  OF  EEPRODUCTION, 

For  Students  of  Human  and  Comparative  Medicine, 
BY  WESLEY  MILLS,  M.  A.,  M.  D., 

PROFESSOR   OF   PHYSIOLOGY  IN    MC  GILL   UNIVERSITY  AND   THE   VETERINARY  COLLEGE,   MONTREAL. 


8vo.    With  505  Illustrations.    Cloth,  $5.00 ;  sheep,  $6.00. 

"...  The  author  has  set  himself  a  task,  as  announced  in  the  preface,  of  trying  to 
make  the  student  an  observer  and  reasoner,  rather  than  merely  to  tax  his  memory ;  to 
acquaint  him  with  the  general  truths  in  the  broad  domain  of  biology,  rather  than  to  over- 
whelm him  with  useless  detail  and  burdensome  statistics.  None  who  carefully  peruse 
his  work  can  fail  to  recognize  that  the  subject  has  been  successfully  presented  in  accord- 
ance with  this  plan.  .  .  .  The  general  merit  of  the  work  easily  places  it  on  a  par  with 
any  text-book  yet  written  for  beginners  in  this  branch  ;  and  the  clear  deductions  of  the 
difference  in  function  and  general  structure  between  man  and  lower  animals  can  not  fail 
to  give  broader  ideas  of  the  whole  science." — JOSEPH  EICHBERG,  M.  D.,  Professor  of 
Physiology  in  Miami  Medical  College,  Cincinnati,  Ohio. 

".  .  .  I  am  pleased  to  accord  this  work  my  hearty  indorsement,  simply  from  the 
fact  that  it  presents  the  subject  in  a  new  way,  and,  strange  to  say,  in  a  manner  that  we 
wonder  had  not  been  thought  of  before,  viz.,  the  comparative  animal  physiology  together 
with  biology  and  embryology,  together  with  evolution,  all  in  a  work  quite  suitable  for  a 
medical  student.  Hitherto  we  have  been  compelled  to  go  to  natural  history  for  these 
matters.  ...  It  surely  deserves  a  place  in  our  literature." — J.  0.  STILLSON,  M.  D.,  Pro- 
fessor of  Physiology  in  the  Central  College  of  Physicians  and  Surgeons  of  Indianapolis,  Ind. 

"  I  am  delighted  with  Dr.  Mills's  book,  the  plan  of  which  is  excellent,  and  the  details 
well  worked  out.  It  will  give  students  in  human  physiology  a  new  insight  into  the  rela- 
tions of  the  subject." — WILLIAM  OSLER,  M.  D.,  Professor  of  Physiology  in  Johns  Hopkins 
University. 

"...  It  fills  a  gap  in  the  works  on  physiology  hitherto  vacant,  and  I  commend  it 
cordially  as  an  excellent  work." — ROBERT  REYBURN,  M.  D.,  Professor  of  Physiology  in  the 
Medical  Department  of  Howard  University,  Washington,  D.  C. 

"  As  a  text-book  for  students  this  work  will  undoubtedly  take  a  high  place,  not  alto- 
gether because  it  is  a  succinct  and  clear  record  of  the  latest  knowledge  in  animal  physi- 
ology, but  also  on  account  of  its  being  founded  on  the  true  principles  of  teaching. 
Especial  care  is  taken  to  point  out  what  is  really  known ;  to  separate  the  known  from 
the  unknown ;  to  show  what  directions  our  investigations  must  take  in  order  that  our 
knowledge  may  increase.  The  work  is  well  printed  and  profusely  illustrated,  and  reflects 
great  credit  on  the  publishers." — Montreal  Medical  Journal. 


New  York :  D.  APPLETON  &  CO..  72  Fifth  Avenue. 


THE 

SCIENCE  AND  AET  OF 

MIDWIFERY. 

BY  WILLIAM  THOMPSON  LUSK,  M.  A.,  M.  D., 

Professor  of  Obstetrics  and  Diseases  of  Women  and  Children  in  the  Bellevue 

Hospital  Medical  College ;  Obstetric  Surgeon  to  the  Maternity 

and  Emergency  Hospitals  ;  and  Gynaecologist 

to  the  Bellevue  Hospital. 


FOURTH   EDITION.     REVISED  AND   REWRITTEN. 
WITH  246  ILLUSTRATIONS. 

8vo.    Cloth,  $5.00;  sheep,  $6.00. 


"  It  was  the  pleasure  of  the  undersigned  to  write  a  review  of  this  most 
excellent  and  masterly  work  on  obstetrics,  when  it  appeared  in  its  first 
edition.  The  present  is  the  fourth,  an  edition  enlarged  and  revised.  It  is 
a  model  of  recent  medical  literature  in  obstetrics,  and  can  not  but  give 
great  credit  to  the  author  and  to  American  medicine,  Model  it  is  of  clear, 
forcible,  and  beautiful  English,  of  good  arrangement  of  subject-matter,  and 
of  thoroughness  of  modern  obstetric  exposition.  The  changes  which  have 
taken  place  in  the  theory  and  practice  of  obstetrics  since  the  issue  of  the 
last  edition  have  made  it  necessary  for  the  author  to  present  to  the  pro- 
fession what  is  essentially  a  new  book.  Most  cheerfully  will  we  recommend 
to  the  students  of  medicine  a  study  of  Lusk.  It  ranks  well  with  Playfair, 
and  is  second  to  no  book  in  our  language." — CHAUNCEY  D.  PALMER,  in  the 
Ohio  Medical  Journal. 

"  The  book  is  now  beyond  criticism,  for  it  has  been  accepted  by  the  un- 
erring judgment  of  the  great  body  of  physicians.  We  congratulate  Dr. 
Lusk  upon  this  reward  for  the  immense  labor  he  has  bestowed  upon  it." — 
New  York  Medical  Journal. 

"It  contains  one  of  the  best  expositions  of  the  obstetric  science  and 
practice  of  the  day  with  which  we  are  acquainted.  Throughout  the  work 
the  author  shows  an  intimate  acquaintance  with  the  literature  of  obstet- 
rics, and  gives  evidence  of  large  practical  experience,  great  discrimination, 
and  sound  judgment.  We  heartily  recommend  the  book  as  a  full  and  clear 
exposition  of  obstetric  science,  and  safe  guide  to  student  and  practitioner." 
— London  Lancet. 

"  It  is  but  a  short  time  since  we  had  occasion  to  review  this  work,  of 
which  we  were  enabled  to  speak  in  the  highest  terms  of  praise.  The  rapid 
advance  of  many  departments  of  obstetrics  has  meantime  called  for  a  few 
additions.  These  having  been  made,  it  can  be  confidently  said  that  Lusk's 
Midwifery  holds  a  high  place  among  American  authors,  and  deserves  to 
be  extensively  employed  for  reference,  and  recommended  to  students  as 
a  reliable  and  unusually  readable  text-book." — Canada  Medical  and 
Surgical  Journal. 


New  York  :  D.  APPLETON'&  CO.,  72  Fifth  Avenue. 


FOURTEEN  DAY  TTSE 

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